Diagnostics and treatments of periodontal disease

ABSTRACT

This invention relates to the PrtR-PrtK cell surface protein of  Porphyromonas gingivalis  in particular a multimeric cell associated protein complex comprising the PrtR and PrtK proteins. There is provided a substantially purified antigenic complex for use in raising an antibody response directed against  Porphyromonas gingivalis.  The complex comprises at least one multimeric protein complex of arginine-specific and lysine-specific thiol endopeptidases each containing at least one adhesin domain. The complex has a molecular weight of greater than about 200 kDa. The invention also relates to pharmaceutical compositions and associated agents based on said complex for the detection, prevention and treatment of Periodontal disease associated with  P. gingivalis.

FIELD OF THE INVENTION

This invention relates to the PrtR-PrtK cell surface protein ofPorphyromonas gingivalis and in particular a multimeric cell associatedprotein complex comprising the PrtR and PrtK proteins. The inventionalso relates to pharmaceutical compositions and associated agents basedon said complex for the detection, prevention and treatment ofPeriodontal disease associated with P. gingivalis.

BACKGROUND OF THE INVENTION

Periodontal diseases are bacterial-associated inflammatory diseases ofthe supporting tissues of the teeth and range from the relatively mildform of gingivitis, the non-specific, reversible inflammation ofgingival tissue to the more aggressive forms of periodontitis which arecharacterised by the destruction of the tooth's supporting structures.Periodontitis is associated with a subgingival infection of a consortiumof specific Gram-negative bacteria that leads to the destruction of theperiodontium and is a major public health problem. One bacterium thathas attracted considerable interest is P. gingivalis as the recovery ofthis microorganism from adult periodontitis lesions can be up to 50% ofthe subgingival anaerobically cultivable flora, whereas P. gingivalis israrely recovered, and then in low numbers, from healthy sites. Aproportional increase in the level of P. gingivalis in subgingivalplaque has been associated with an increased severity of periodontitisand eradication of the microorganism from the cultivable subgingivalmicrobial population is accompanied by resolution of the disease. Theprogression of periodontitis lesions in non-human primates has beendemonstrated with the subgingival implantation of P. gingivalis. Thesefindings in both animals and humans suggest a major role for P.gingivalis in the development of adult periodontitis.

P. gingivalis is a black-pigmented, anaerobic, asaccharolytic,proteolytic Gram-negative rod that obtains energy from the metabolism ofspecific amino acids. The microorganism has an absolute growthrequirement for iron, preferentially in the form of haeme or its Fe(III)oxidation product haemin and when grown under conditions of excesshaemin is highly virulent in experimental animals. A number of virulencefactors have been implicated in the pathogenicity of P. gingivalisincluding the capsule, adhesins, cytotoxins and extracellular hydrolyticenzymes. In particular, proteases have received a great deal ofattention for their ability to degrade a broad range of host proteinsincluding structural proteins and others involved in defence. Theproteins that have been shown to be substrates for P. gingivalisproteolytic activity include collagen types I and IV, fibronectin,fibrinogen, laminin, complement and plasma clotting cascade proteins,α₁-antitrypsin, α₂-macroglobulin, antichymotrypsin, antithrombin III,antiplasmin, cystatin C, IgG and IgA. The major proteolytic activitiesassociated with this organism have been defined by substrate specificityand are “trypsin-like”, that is cleavage on the carboxyl side of arginyland lysyl residues and collagenolytic although other minor activitieshave been reported.

P. gingivalis trypsin-like proteolytic activity has been shown todegrade complement, generating biologically active C5a, impair thephagocytic and other functions of neutrophils by modifying surfacereceptors, and abrogate the clotting potential of fibrinogen prolongingplasma clotting time. The trypsin-like proteolytic activity of P.gingivalis also generates Fc fragments from human IgG1 stimulating therelease of pro-inflammatory cytokines from mononuclear cells and isassociated with vascular disruption and enhanced vascular permeationthrough the activation of the kallikrein-kinin cascade. P. gingivalisspontaneous mutants with reduced trypsin-like activity as well aswild-type cells treated with the trypsin-like protease inhibitorN-p-tosyl-L-lysine chloromethyl ketone are avirulent in animal models.Further, it has been shown that P. gingivalis grown under controlled,haemin-excess conditions expressed more trypsin-like and lesscollagenolytic activity and were more virulent in mice relative to cellsgrown under haemin-limited but otherwise identical conditions. Theincreased expression of the trypsin-like activity by the more virulentP. gingivalis has led to the speculation that the trypsin-likeproteolytic activity may be the major determinant for infection ordisease. However, the cell-associated trypsin-like proteolyticactivities of P. gingivalis have not been characterised to date.

There has been considerable endeavour to purify and characterise thetrypsin-like proteases of P. gingivalis from cell-free culture fluids.Chen et al, (1992) [J Biol Chem 267:18896-18901] have purified andcharacterised a 50 kDa arginine-specific, thiol protease from theculture fluid of P. gingivalis H66 designated Arg-gingipain. A similararginine-specific thiol protease has been disclosed in JP 07135973 andthe amino acid sequence disclosed in WO 9507286 and in Kirszbaum et al,1995 [Biochem Biophys Res Comm 207:424-431]. Pike et al (1994) [J BiolChem 269:406-411] have characterised a 60 kDa lysine-specific cysteineproteinase from the culture fluid of P. gingivalis H66 designatedLys-gingipain and the partial gene sequence for this enzyme wasdisclosed in WO 9511298 and fully disclosed in WO 9617936. However,prior to the development of the present invention it was unknown thatthere existed on the cell surface of P. gingivalis a 300 kda complex ofarginine-specific and lysine-specific proteases both containing adhesindomains. The 300 kDa complex has been designated the PrtR-PrtK complex.The presence of the PrtR-PrtK cell surface complex exhibiting botharginine- and lysine-specific proteolytic activity together with adhesinactivity was previously unknown. Furthermore, the new PrtR-PrtK complexof the present invention is expressed on the cell surface, is a majorvirulence-associated factor and contains unique epitopes not displayedon the individual domains. The previously disclosed arginine-specificand lysine-specific thiol proteases, as discussed, do not exhibit any ofthese features and have proven of limited application to date. However,the aforementioned features have rendered the PrtR-PrtK complex of theinvention ideal for development of diagnostic and immunoprophylacticproducts. The PrtR-PrtK cell surface complex is accordingly ofparticular interest for diagnostics and neutralisation by passiveimmunity through oral compositions containing neutralising antibodiesand by vaccine development. In particular for the development of anintra-oral recombinant bacterial vaccine, where the recombinantbacterium expressing an inactivated PrtR-PrtK is a geneticallyengineered commensal inhabitant of the oral cavity.

SUMMARY OF THE INVENTION

Accordingly in a first aspect the present invention consists in asubstantially purified antigenic complex for use in raising an antibodyresponse directed against Porphyromonas gingivalis, the complexcomprising at least one multimeric protein complex of arginine-specificand lysine-specific thiol endopeptidases each containing at least oneadhesin domain, the complex having a molecular weight of greater thanabout 200 kDa.

In the context of this disclosure, the terms “adhesin” and“hemagglutinin” may be considered to be synonymous.

In a preferred form of the present invention the multimeric proteincomplex is associated with virulent strains of Porphyromonas gingivalis,preferably has a molecular weight of about 294 to about 323 kDa and ispreferably derived from P. gingivalis W50.

It is also preferred that the multimeric protein complex is composed of9 proteins. These 9 proteins preferably have the following N-terminalsequences:

DVYTDHGDLYNTPVRML (SEQ ID NO: 1)

YTPVEEKQNGRMIVIVAKKYEGD (SEQ ID NO: 2)

SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHFL (SEQ ID NO: 3)

PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO: 4)

ANEAKVVLAADNVWGDNTGYQFLLDA (SEQ ID NO: 5)

ANEAKVVLAADNVWGDNTGYQFLLDA (SEQ ID NO: 5)

PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO: 4)

ADFTETFESSTHGEAPAEWTTIDA (SEQ ID NO: 6)

ADFTETFESSTHGEAPAEWTTIDA. (SEQ ID NO: 6)

It is presently preferred that the 9 proteins are PrtK48, PrtR45,PrtR44, PrtK39, PrtK44, PrtR27, PrtR17, PrtK15 and PrtR15 as describedherein.

As the purified antigenic complex normally has enzymatic activity it ispreferred in a number of uses the thiol endopeptidases are renderedinactive. This may be achieved in a number of ways, for example byoxidation or by mutation. It is presently preferred that theinactivation is by oxidation.

In yet another preferred embodiment of the present invention themultimeric protein complex is encoded by the DNA sequence shown in FIGS.8B and 9B.

In a second aspect the present invention consists in a composition foruse in eliciting an immune response directed against Porphyromonasgingivalis, the composition comprising an effective amount of thecomplex of the first aspect of the present invention and a suitableadjuvant and/or acceptable carrier.

In a third aspect the present invention consists in an antibodypreparation comprising antibodies specifically directed against thecomplex of the first aspect of the present invention. The antibodies maybe polyclonal antibodies or monoclonal antibodies.

In a fourth aspect the present invention consists in a method oftreating a subject suffering from Porphyromonas gingivalis infection,the method comprising administering to the subject an amount of theantibody preparation of the third aspect of the present inventioneffective to at least partially neutralize the PrtR-PrtK complex ofPorphyromonas gingivalis.

As will be recognised by those skilled in the art the antibodypreparation may be administered by any of a number of well known routes,however, it is presently preferred that the preparation is administeredorally.

In a fifth aspect the present invention consists in a method of reducingthe prospect of P. gingivalis infection in an individual and/or severityof disease, the method comprising administering to the individual anamount of the composition of the second aspect of the present inventioneffective to induce an immune response in the individual directedagainst P. gingivalis.

In yet a further aspect the present invention consists in a recombinanthost cell, the host cell being transformed with a DNA sequence(s)encoding PrtR-PrtK operatively linked to control sequences such thatunder appropriate conditions the host cell expresses PrtR-PrtK.

In another aspect, the present invention is directed to novel DNAsequences involving PrtR-PrtK constructs and vectors including plasmidDNA, and viral DNA such as human viruses, animal viruses, insectviruses, or bacteriophages which can be used to direct the expression ofPrtR-PrtK protein in appropriate host cells from which the expressedprotein may be purified. Another aspect of the present inventionprovides methods for molecular cloning of the genes encoding thePrtR-PrtK complex. The nucleic acid sequences of the present inventioncan be used in molecular diagnostic assays for P. gingivalis geneticmaterial through nucleic acid hybridization, and including the synthesisof PrtR-PrtK sequence-specific oligonucleotides for use as primersand/or probes in amplifying, and detecting amplified, nucleic acids.Additionally, PrtR-PrtK complex can be used as an immunogen inprophylactic and/or therapeutic vaccine formulations against pathogenicstrains of P. gingivalis, whether the immunogen is chemicallysynthesized, purified from P. gingivalis, or purified from a recombinantexpression vector system. Alternatively, the genes encoding PrtR-PrtKmay be incorporated into a bacterial or viral vaccine comprisingrecombinant bacteria or virus which is engineered to produce PrtR-PrtKby itself, or in combination with immunogenic epitopes of otherpathogenic microorganisms. In addition, the genes encoding PrtR-PrtKoperatively linked to one or more regulatory elements, can be introduceddirectly into humans to express the PrtR-PrtK to elicit a protectiveimmune response. A vaccine can also be based upon a recombinantcomponent of a mutated PrtR-PrtK incorporated into an appropriate vectorand expressed in a suitable transformed host (eg. E. coli, Bacillussubtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells)containing the vector. The vaccine can be based on an intra-oralrecombinant bacterial vaccine, where the recombinant bacteriumexpressing an inactivated PrtR-PrtK is a commensal inhabitant of theoral cavity. Unlike whole P. gingivalis cells or other previouslyprepared antigens based on fimbriae or the capsule the PrtR-PrtK complexof the invention or component parts thereof are safe and effectiveantigens for the preparation of a vaccine for the prevention of P.gingivalis-associated periodontal disease. The invention thereforeprovides a range of recombinant products based on the PrtR-PrtK complex.

The invention also provides antibodies raised against the said PrtR-PrtKcomplex, herein called anti-PrtR-PrtK antibodies. The antibodies may beblended into oral compositions such as toothpaste, mouthwash,toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums,dental pastes, gingival massage creams, gargle tablets, dairy productsand other foodstuffs.

In another aspect the invention provides a method of diagnosis for thepresence of P. gingivalis characterised by the use of any one or acombination of an antibody, antigen or nucleic acid probe ashereinbefore defined comprising the application of known techniquesincluding for example, enzyme linked immunosorbent assay.

The invention also provides diagnostic kits comprising antibodies,antigens and/or nucleic acid probes as hereinbefore defined.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Anion exchange FPLC of a P. gingivalis W50 sonicate. Thesonicate in TC buffer containing 50 mM NaCl was applied to a Hiload XK16/10 Q sepharose column and eluted using a linear gradient from 0-100%buffer B over 90 min at a flow rate of 2.0 ml min⁻¹. Fractions (6 ml)were assayed for proteolytic and amidolytic activity using azocasein,Bz-L-Arg-pNA and Z-L-Lys-pNA. The amidolytic activity of each 6 mlfraction with Bz-L-Arg-pNA is shown by the histogram.

FIG. 2. Gel filtration FPLC of the pooled and concentrated fractionsfrom Q sepharose anion exchange FPLC containing proteolytic/amidolyticactivity. Anion exchange fractions containing the major peak ofproteolytic/amidolytic activity were pooled, equilibrated in TC buffercontaining 150 mM NaCl, concentrated and divided into four aliquots andeach then independently applied to a gel filtration column (Superose 12HR 10/30) and eluted using the same buffer at a flow rate of 0.3 mlmin⁻¹. Fractions (0.5 ml) were assayed for proteolytic and amidolyticactivity. Bz-L-Arg-pNA amidolytic activity is shown by the histogram. Voand Vt indicate the void and total volumes of the column respectively.The elution volumes of the standard proteins thyroglobulin 667 kDa,catalase 232 kDa and aldolase 158 kDa are marked.

FIG. 3. SDS-PAGE (boiled/reduced conditions) of the 300 kDa peak fromgel filtration (Superose 12 HR 10/30) FPLC. Lane 1, Pharmacia molecularmass standards (M_(r) shown in kDa). Lane 2, 300 kDa peak from gelfiltration FPLC. Coomassie blue stained gel.

FIG. 4. Specific cleavage sites (marked with arrows) of α_(s1)-casein bythe proteolytic/amidolytic peak from gel filtration FPLC correspondingto 300 kDa. The protein α_(s1)-casein was cleaved on the carboxyl sideof arginyl and lysyl residues only.

FIG. 5. Arg-sepharose FPLC of the 300 kDa gel filtration peak exhibitingArg- and Lys-specific proteolytic activity. Gel filtration fractionscontaining the major peak of proteolytic activity (300 kDa) were pooledand applied to an arginine-sepharose column (5 ml arginine-Sepharose 4B)and washed with TC buffer containing 50 mM NaCl at 0.1 ml min⁻¹until thebaseline returned to zero. The column was then further washed with 500mM NaCl and then re-equilibrated with TC buffer containing 50 mM NaCl.The column was first eluted with 200 mM lysine in TC buffer containing50 mM NaCl, followed by 750 mM lysine in the same buffer. The column wasthen re-equilibrated and eluted with 200 mM arginine in the same bufferat a flow rate of 0.1 ml min⁻¹. Peaks were collected and assayed foramidolytic and proteolytic activity. Bz-L-Arg-pNA amidolytic activity isshown by the histogram and the arrows indicate the start of each stepgradient.

FIG. 6. SDS-PAGE (boiled/reduced conditions) of 200 mM lysine eluantfrom the Arg-sepharose FPLC. Lane 1, Pharmacia molecular mass standards(M, shown in kDa). Lane 2, 200 mM lysine eluant from Arg-sepharose FPLC.Silver stained gel.

FIG. 7. SDS-PAGE (boiled/reduced conditions) of the 750 mM lysine and200 mM arginine eluants from the arginine-Sepharose FPLC and thepurified 45 kDa Arg-specific endopeptidase. Lane 1, 750 mM lysineeluant. Lane 2, 200 mM arginine eluant. Lane 3, purified 45 kDaArg-specific endopeptidase. Lane 4, Pharmacia molecular mass standards(M_(r) shown in kDa). Coomassie blue stained gel.

FIG. 8a. Schematic representation of the prtR gene. The PrtR nascentpolyprotein is composed of a leader sequence, a prosequence followed bythe Arg-specific cysteine proteinase PrtR45 (SEQ ID NO: 2, residues1-16), and the adhesins PrtR44 (SEQ ID NO: 3, residues 1-16), PrtR15(SEQ ID NO: 6, residues 1-16), PrtR17 (SEQ ID NO: 4, residues 1-16) andPrtR27 (SEQ ID NO: 5, residues 1-16) all preceded by an arginyl or lysylresidue.

FIG. 8b-1-8 b-4. Nucleotide sequence of prtR (SEQ ID NO: 7).

FIG. 9a. Schematic representation of the prtK gene. The PrtK nascentpolyprotein is composed of a leader sequence, a prosequence followed bythe Lys-specific cysteine proteinase PrtK48 (SEQ ID NO: 1), and theadhesins PrtK39 (SEQ ID NO: 5, residues 1-16), PrtK15 (SEQ ID NO: 6,residues 1-16), and PrtK44 (SEQ ID NO: 4, residues 1-16), all precededby an arginyl or lysyl residue.

FIG. 9b-1-9 b-3. Nucleotide sequence of prtK (SEQ ID NO: 8).

FIG. 10. SDS-PAGE of the PrtR-PrtK complex purified by diafiltration.Lane 1 shows molecular mass markers. Lane 2 shows components of thePrtR-PrtK purified by diafiltration.

FIG. 11. ELISA titration of sera from 5 mice immunized twice with thePrtR-PrtK complex emulsified in Freund's Incomplete Adjuvant. Test sera(TS 32-36) and pre-immune sera (PIS 32-36) were screened using P.gingivalis W50 sonicate as the adsorbed antigen. Primary antibodydilutions of 1/100, 1/500, 1/2500 and 1/12500 were used. Bound antibodywas determined using horseradish peroxidase-conjugated goat anti-mouseantibody and 3,3′,5,5′ tetramethylbenezidine. The reaction product wasquantitated spectrophotometrically using a 450 mn interference filter ina plate reader and recorded as optical density (O.D.) readings.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in greater detail by reference tothe methods used and applied in the development of the invention and byreference to particular examples which provide the best methods known ofperforming the invention.

The intra-oral bacterium Porphyromonas gingivalis possesses on its cellsurface major trypsin-like proteinases as a 294-323 kDa heterodimericprotein complex of Arg-specific and Lys-specific thiol endopeptidaseswith hemagglutinins (adhesins) herein designated the PrtR-PrtK complex.The PrtR-PrtK complex can be purified from P. gingivalis cells byultrasonication or chloroform extraction followed by diafiltration oranion exchange and Lys-sepharose or Arg-sepharose chromatography. Thepurified PrtR-PrtK complex is then used to generate antibodies usingstandard techniques. The animals used for antibody generation can berabbits, goats, chickens, sheep, horses, cows etc. When a high antibodytitre against the PrtR-PrtK complex is detected by immunoassay theanimals are bled or eggs or milk are collected and the serum preparedand/or antibody purified using standard techniques or monoclonalantibodies produced by fusing spleen cells with myeloma cells usingstandard techniques. The antibody (immunoglobulin fraction) may beseparated from the culture or ascites fluid, serum, milk or egg bysalting out, gel filtration, ion exchange and/or affinitychromatography, and the like, with salting out being preferred. In thesalting out method the antiserum or the milk is saturated with ammoniumsulphate to produce a precipitate, followed by dialyzing the precipitateagainst physiological saline to obtain the purified immunoglobulinfraction with the specific anti-(PrtR-PrtK). The preferred antibody isobtained from the equine antiserum and the bovine antiserum and milk. Inthis invention the antibody contained in the antiserum and milk obtainedby immunising the animal with the inactivated PrtR-PrtK may be blendedinto the oral composition. In this case the antiserum and milk as wellas the antibody separated and purified from the antiserum and milk maybe used. Each of these materials may be used alone or in combination oftwo or more. Antibodies against the PrtR-PrtK can be used in oralcompositions such as toothpaste and mouthwash to neutralise thePrtR-PrtK and thus prevent disease. The anti-(PrtR-PrtK) antibodies canalso be used for the early detection of P. gingivalis in subgingivalplaque samples by a chairside Enzyme Linked Immunosorbent Assay (ELISA).

For oral compositions it is preferred that the amount of the aboveantibodies administered is 0.0001-50 g/kg/day and that the content ofthe above antibodies is 0.0002-10% by weight preferably 0.002-5% byweight of the composition. The oral composition of this invention whichcontains the above-mentioned serum or milk antibody may be prepared andused in various forms applicable to the mouth such as dentifriceincluding toothpastes, toothpowders and liquid dentifrices, mouthwashes,troches, periodontal pocket irrigating devices, chewing gums, dentalpastes, gingival massage creams, gargle tablets, dairy products andother foodstuffs. The oral composition according to this invention mayfurther include additional well known ingredients depending on the typeand form of a particular oral composition.

In certain highly preferred forms of the invention the oral compositionmay be substantially liquid in character, such as a mouthwash or rinse.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humectant as described below. Generally, theweight ratio of water to alcohol is in the range of from about 1:1 toabout 20:1. The total amount of water-alcohol mixture in this type ofpreparation is typically in the range of from about 70 to about 99.9% byweight of the preparation. The alcohol is typically ethanol orisopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 4.5 to about 9 and typically fromabout 5.5 to 8. The pH is preferably in the range of from about 6 toabout 8.0, preferably 7.4. The pH can be controlled with acid (e.g.citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered(as with sodium citrate, benzoate, carbonate, or bicarbonate, disodiumhydrogen phosphate, sodium dihydrogen phosphate, etc).

Other desirable forms of this invention, the oral composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a dentifrice, that is a toothpaste (dental cream) or geldentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material. Examples ofpolishing materials are water-insoluble sodium metaphosphate, potassiummetaphosphate, tricalcium phosphate, dihydrated calcium phosphate,anhydrous dicalcium phosphate, calcium pyrophosphate, magnesiumorthophosphate, trimagnesium phosphate, calcium carbonate, hydratedalumina, calcined alumina, aluminium silicate, zirconium silicate,silica, bentonite, and mixtures thereof. Other suitable polishingmaterial include the particulate thermosetting resins such as melamine-,phenolic, and urea-formaldehydes, and cross-linked polyepoxides andpolyesters. Preferred polishing materials include crystalline silicahaving particle sized of up to about 5 microns, a mean particle size ofup to about 1.1 microns, and a surface area of up to about 50,000cm²/gm., silica gel or colloidal silica, and complex amorphous alkalimetal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metalalumino-silicate complexes are particularly useful since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner asillustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4thEdition, pp. 510-511. The forms of insoluble sodium metaphosphate knownas Madrell's salt and Kurrol's salt are further examples of suitablematerials. These metaphosphate salts exhibit only a minute solubility inwater, and therefore are commonly referred to as insolublemetaphosphates (IMP). There is present therein a minor amount of solublephosphate material as impurities, usually a few percent such as up to 4%by weight. The amount of soluble phosphate material, which is believedto include a soluble sodium trimetaphosphate in the case of insolublemetaphosphate, may be reduced or eliminated by washing with water ifdesired. The insoluble alkali metal metaphosphate is typically employedin powder form of a particle size such that no more than 1% of thematerial is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight. Other polishingmaterials are typically present in amount of about 30-75% by weight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5-30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationsare to be sold or otherwise distributed in suitable labelled packages.Thus, ajar of mouthrinse will have a label describing it, in substance,as a mouthrinse or mouthwash and having directions for its use; and atoothpaste, cream or gel will usually be in a collapsible tube,typically aluminium, lined lead or plastic, or other squeeze, pump orpressurised dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature which does notdenature the antibody of the invention, and it is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties while not denaturing theantibody. Suitable examples of anionic surfactants are water-solublesalts of higher fatty acid monoglyceride monosulfates, such as thesodium salt of the monosulfated monoglyceride of hydrogenated coconutoil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate,alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higheralkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propanesulfonate, and the substantially saturated higher aliphatic acyl amidesof lower aliphatic amino carboxylic acid compounds, such as those having12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and thelike. Examples of the last mentioned amides are N-lauroyl sarcosine, andthe sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl,or N-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants suitable for use with antibodies are condensation productsof ethylene oxide with various reactive hydrogen-containing compoundsreactive therewith having long hydrophobic chains (e.g. aliphatic chainsof about 12 to 20 carbon atoms), which condensation products(“ethoxamers”) contain hydrophilic polyoxyethylene moieties, such ascondensation products of poly (ethylene oxide) with fatty acids, fattyalcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate)and polypropyleneoxide (e.g. Pluronic materials).

Surface active agent is typically present in amount of about 0.1-5% byweight. It is noteworthy, that the surface active agent may assist inthe dissolving of the antibody of the invention and thereby diminish theamount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavouring or sweetening material may also be employed.Examples of suitable flavouring constituents are flavouring oils, e.g.oil of spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavour and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 5.5 to about 8, preferably about 6 to 8, for at least 2weeks up to 8 weeks or more up to a lifetime.

The compositions of this invention can be incorporated in lozenges, orin chewing gum or other products, e.g. by stirring into a warm gum baseor coating the outer surface of a gum base, illustrative of which may bementioned jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticisers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like.

The composition of this invention also includes targeted deliveryvehicles such as periodontal pocket irrigation devices, collagen,elastin, or synthetic sponges, membranes or fibres placed in theperiodontal pocket or used as a barrier membrane or applied directly tothe tooth root.

Another important form of the invention is a composition for use ineliciting an immune response directed against Porphyromonas gingivalisbased on the PrtR-PrtK complex and suitable adjuvant delivered by nasalspray, orally or by injection to produce a specific immune responseagainst the PrtR-PrtK complex thereby reducing colonisation of P.gingivalis and neutralising the PrtR-PrtK thereby preventing disease.Due to the potent enzymatic activity of the complex typically thecomplex will be inactivated. A vaccine can also be based upon arecombinant component of the PrtR-PrtK incorporated into an appropriatevector and expressed in a suitable transformed host (eg. E. coli,Bacillus subtilis, Saccharomyces cerevisiae, COS cells, CHO cells andHeLa cells) containing the vector. Unlike whole P. gingivalis cells orother previously prepared antigens based on fimbriae or the capsule thePrtR-PrtK complex is a safe and effective antigens for the preparationof a composition for use in the prevention of P. gingivalis-associatedperiodontal disease. The PrtR-PrtK complex can be produced usingrecombinant DNA methods as illustrated herein, or can be synthesizedchemically from the amino acid sequence disclosed in the presentinvention. Additionally, according to the present invention, thePrtR-PrtK complex may be used to generate antisera useful for passiveimmunization against periodontal disease and infections caused by P.gingivalis.

Various adjuvants are used in conjunction with vaccine formulations. Theadjuvants aid by modulating the immune response and in attaining a moredurable and higher level of immunity using smaller amounts of vaccineantigen or fewer doses than if the vaccine antigen were administeredalone. Examples of adjuvants include incomplete Freund's adjuvant (IFA),Adjuvant 65 (containing peanut oil, mannide monooleate and aluminiummonostrearate), oil emulsions, Ribi adjuvant, the pluronic polyols,polyamines, Avridine, Quil A, saponin, MPL, QS-21, and mineral gels suchas aluminium salts. Other examples include oil in water emulsions suchas SAF-1, SAF-0, MF59, Seppic ISA720, and other particulate adjuvantssuch as ISCOMs and ISCOM matrix. An extensive but not exhaustive list ofother examples of adjuvants are listed in Cox and Coulter 1992 [In: WongW K (ed.) Animals parasite control utilising technology. Bocca Raton;CRC press, 1992; 49-112]. In addition to the adjuvant the vaccine mayinclude conventional pharmaceutically acceptable carriers, excipients,fillers, buffers or diluents as appropriate. One or more doses of thevaccine containing adjuvant may be administered prophylactically toprevent periodontitis or therapeutically to treat already presentperiodontitis.

In another preferred composition the preparation is combined with amucosal adjuvant and administered via the oral route. Examples ofmucosal adjuvants are cholera toxin and heat labile E. coli toxin, thenon-toxic B subunits of these toxins, genetic mutants of these toxinswhich have a reduced toxicity. Other methods which may be utilised todeliver the PrtR-PrtK complex orally include incorporation of theprotease into particles of biodegradable polymers (such as acrylates orpolyesters) by microencapsulation to aid uptake of the microspheres fromthe gastrointestinal tract and to protect degradation of the proteins.Liposomes, ISCOMs, hydrogels are examples of other potential methodswhich may be further enhanced by the incorporation of targetingmolecules such as LTB, CTB or lectins for delivery of the PrtR-PrtKcomplex to the mucosal immune system. In addition to the vaccine and themucosal adjuvant or delivery system the vaccine may include conventionalpharmaceutically acceptable carriers, excipients, fillers, coatings,dispersion media, antibacterial and antifungal agents, buffers ordiluents as appropriate.

Another mode of this embodiment provides for either a live recombinantviral vaccine, recombinant bacterial vaccine, recombinant attenuatedbacterial vaccine, or an inactivated recombinant viral vaccine which isused to protect against infections caused by P. gingivalis. Vacciniavirus is the best known example, in the art, of an infectious virus thatis engineered to express vaccine antigens derived from other organisms.The recombinant live vaccinia virus, which is attenuated or otherwisetreated so that it does not cause disease by itself is used to immunizethe host. Subsequent replication of the recombinant virus within thehost provides a continual stimulation of the immune system with thevaccine antigens such as PrtR-PrtK complex, thereby providing longlasting immunity.

Other live vaccine vectors include: adenovirus, cytomegalovirus, andpreferably the poxviruses such as vaccinia (Paoletti and Panicali, U.S.Pat. No. 4,603,112) and attenuated Salmonella strains (Stocker et al.,U.S. Pat. Nos. 5,210,035; 4,837,151; and 4,735,801; and Curtiss et al.,1988, Vaccine 6:155-160). Live vaccines are particularly advantageousbecause they continually stimulate the immune system which can confersubstantially long-lasting immunity. When the immune response isprotective against subsequent P. gingivalis infection, the live vaccineitself may be used in a preventive vaccine against P. gingivalis. Inparticular, the live vaccine can be based on a bacterium that is acommensal inhabitant of the oral cavity. This bacterium can betransformed with a vector carrying a recombinant inactivated PrtR-PrtKand then used to colonise the oral cavity, in particular the oralmucosa. Once colonised the oral mucosa, the expression of therecombinant protein will stimulate the mucosal associated lymphoidtissue to produce neutralising antibodies. For example, using molecularbiological techniques the genes encoding the PrtR-PrtK may be insertedinto the vaccinia virus genomic DNA at a site which allows forexpression of epitopes but does not negatively affect the growth orreplication of the vaccinia virus vector. The resultant recombinantvirus can be used as the immunogen in a vaccine formulation. The samemethods can be used to construct an inactivated recombinant viralvaccine formulation except that the recombinant virus is inactivated,such as by chemical means known in the art, prior to use as an immunogenand without substantially affecting the immunogenicity of the expressedimmunogen.

In another variation of this embodiment, genetic material is useddirectly as the vaccine formulation. Nucleic acid (DNA or RNA)containing sequences encoding the PrtR-PrtK protein complex operativelylinked to one or more regulatory elements can be introduced directly tovaccinate the individual (“direct gene transfer”) against pathogenicstrains of P. gingivalis. Direct gene transfer into a vaccinatedindividual, resulting in expression of the genetic material by thevaccinated individual's cells such as vascular endothelial cells as wellas the tissue of the major organs, has been demonstrated by techniquesin the art such as by injecting intravenously an expressionplasmid:cationic liposome complex (Zhu et al., 1993, Science261:209-211). Other effective methods for delivering vector DNA into atarget cell are known in the art. In one example, purified recombinantplasmid DNA containing viral genes has been used to inoculate (whetherparentally, mucosally, or via gene-gun immunization) vaccines to inducea protective immune response (Fynan et al. 1993, Proc. Natl. Acad. Sci.USA 90:11478-11482). In another example, cells removed from anindividual can be transfected or electroporated by standard proceduresknown in the art, resulting in the introduction of the recombinantvector DNA into the target cell. Cells containing the recombinant vectorDNA may then be selected for using methods known in the art such as viaa selection marker expressed in the vector, and the selected cells maythen be re-introduced into the individual to express the PrtR-PrtKcomplex.

As an alternative to active immunization, immunization may be passive,i.e. immunization comprising administration of purified immunoglobulincontaining antibody against PrtR-PrtK.

The present invention further provides the nucleotide sequence of thegenes encoding the PrtR-PrtK complex, as well as the amino acid sequencededuced from the isolated genes. According to one embodiment of thepresent invention, using recombinant DNA techniques the genes encodingthe PrtR-PrtK complex are incorporated into an expression vector, andthe recombinant vector is introduced into an appropriate host cellthereby directing the expression of these sequences in that particularhost cell. The expression system, comprising the recombinant vectorintroduced into the host cell, can be used (a) to produce PrtR-PrtKcomplex which can be purified for use as an immunogen in vaccineformulations; (b) to produce PrtR-PrtK complex to be used as an antigenfor diagnostic immunoassays or for generating P. gingivalis-specificantisera of therapeutic and/or diagnostic value; (c) or if therecombinant expression vector is a live virus such as vaccinia virus,the vector itself may be used as a live or inactivated vaccinepreparation to be introduced into the host's cells for expression ofPrtR-PrtK complex; (d) for introduction into live attenuated bacterialcells or genetically engineered commensal intra-oral bacteria which areused to express PrtR-PrtK complex to vaccinate individuals; (e) or forintroduction directly into an individual to immunize against the encodedand expressed PrtR-PrtK complex. In particular the recombinant bacterialvaccine can be based on a commensal inhabitant of the human oral cavityor animal if the vaccine is to prevent periodontal disease in animals.The recombinant bacterial vaccine expressing inactivated PrtR-PrtK canbe used to colonise the oral cavity, supragingival or subgingivalplaque. The intra-oral bacterium can be isolated from the patient withperiodontitis and genetically engineered to express inactivatedPrtR-PrtK complex. The production of the inactivated PrtR-PrtK withinthe oral cavity will not be toxic to the oral mucosal tissues. However,the inactivated PrtR-PrtK will stimulate the mucosal-associated lymphoidtissues (MALT) to produce specific antibody to neutralise the PrtR-PrtKof P. gingivalis.

Successful expression of a protein or peptide requires that either theinsert comprising the gene or gene fragment, or the vector itself,contain the necessary elements for transcription and translation whichis compatible with, and recognized by the particular host system usedfor expression. A variety of host systems may be utilized to express thePrtR-PrtK, which include, but are not limited to bacteria transformedwith a bacteriophage vector, plasmid vector, or cosmid DNA, yeastcontaining yeast vectors; fungi containing fungal vectors; insect celllines infected with virus (e.g. baculovirus); and mammalian cell linestransfected with plasmid or viral expression vectors, or infected withrecombinant virus (e.g. vaccinia virus, adenovirus, adeno-associatedvirus, retrovirus, etc.).

Using methods known in the art of molecular biology various promotersand enhancers can be incorporated into the vector or the DNA sequenceencoding PrtR-PrtK to increase the expression of the PrtR-PrtK aminoacid sequences, provided that the increased expression of the amino acidsequences is compatible with (for example, non-toxic to) the particularhost cell system used. Further, the DNA can be fused to DNA encodingother antigens, such as other bacterial outer membrane proteins, orother bacterial, fungal, parasitic, or viral antigens to create agenetically fused (sharing a common peptide backbone) multivalentantigen for use as an improved vaccine composition.

The selection of the promoter will depend on the expression system used.Promoters vary in strength, i.e. ability to facilitate transcription.Generally, for the purpose of expressing a cloned gene, it is desirableto use a strong promoter in order to obtain a high level oftranscription of the gene and expression into gene product. For example,bacterial, phage, or plasmid promoters known in the art from which ahigh level of transcription have been observed in a host cell systemcomprising E. coli include the lac promoter, trp promoter, recApromoter, ribosomal RNA promoter, the P_(R) and P_(L) promoters, lacUV5,ompF, bla, lpp, and the like, may be used to provide transcription ofthe inserted DNA sequence encoding PrtR-PrtK.

Additional, if PrtR-PrtK protein may be lethal or detrimental to thehost cells, the host cell strain/line and expression vectors may bechosen such that the action of the promoter is inhibited untilspecifically induced. For example, in certain operons the addition ofspecific inducers is necessary for efficient transcription of theinserted DNA (e.g., the lac operon is induced by the addition of lactoseor isopropylthio-beta-D-galactoside). A variety of operons such as thetrp operon, are under different control mechanisms. The trp operon isinduced when tryptophan is absent in the growth media. The PL promotercan be induced by an increase in temperature of host cells containing atemperature sensitive lambda repressor. In this way, greater than 95% ofthe promoter-directed transcription may be inhibited in uninduced cells.Thus, expression of recombinant PrtR-PrtK protein may be controlled byculturing transformed or transfected cells under conditions such thatthe promoter controlling the expression from the inserted DNA encodingPrtR-PrtK amino acid sequences is not induced, and when the cells reacha suitable density in the growth medium, the promoter can be induced forexpression from the inserted DNA.

Other control elements for efficient gene transcription or messagetranslation include enchancers, and regulatory signals. Enhancersequences are DNA elements that appear to increase transcriptionalefficiency in a manner relatively independent of their position andorientation with respect to a nearby gene. Thus, depending on the hostcell expression vector system used, an enhancer may be placed eitherupstream or downstream from the inserted DNA sequences encodingPrtR-PrtK amino acid sequences to increase transcriptional efficiency.These or other regulatory sites, such as transcription or translationinitiation signals, can be used to regulate the expression of the geneencoding PrtR-PrtK. Such regulatory elements may be inserted into DNAsequences encoding PrtR-PrtK amino acid sequences or nearby vector DNAsequences using recombinant DNA methods described herein for insertionof DNA sequences.

Accordingly, P. gingivalis nucleotide sequences containing regionsencoding for PrtR-PrtK, can be ligated into an expression vector at aspecific site in relation to the vectors promoter, control, andregulatory elements so that when the recombinant vector is introducedinto the host cell the P. gingivalis-specific DNA sequences can beexpressed in the host cell. For example, the PrtR-PrtK specific DNAsequences containing their own regulatory elements can be ligated intoan expression vector in a relation or orientation to the vector promoterand control elements which will allow for co-expression of the PrtR andPrtK. The recombinant vector is then introduced into the appropriatehost cells, and the host cells are selected, and screened for thosecells containing the recombinant vector. Selection and screening may beaccomplished by methods known in the art including detecting theexpression of a marker gene (e.g., drug resistance marker) present inthe plasmid, immunoscreening for production of PrtR-PrtK specificepitopes using antisera generated to PrtR-PrtK specific epitopes, andprobing the DNA of the host's cells for PrtR-PrtK specific nucleotidesequence using one or more oligonucleotides and methods describedherein.

Genetic engineering techniques may also be used to characterize, modifyand/or adapt the encoded PrtR-PrtK protein. For example, site-directedmutagenesis to inactivate the protease domains of the PrtR-PrtK and tomodify the protein in regions outside the protective domains, may bedesirable to increase the safety and solubility.

In particular the host organism for the vector containing the PrtR-PrtKgenes and constructs can be a commensal inhabitant of the oral cavity;for example an inhabitant of subgingival plaque, supragingival plaque ora bacterium associated with the oral mucosa. Examples of commensalintra-oral bacteria would be Streptococcus species and Actinomycesspecies, eg. Streptococcus salivarius, Streptococcas sanguis,Actinomyces naeslundii. These organisms can be isolated from theperiodontitis patient and then genetically engineered to express theinactivated PrtR-PrtK. The DNA encoding the PrtR-PrtK could be linkedwith DNA encoding leader sequences of extracellular proteins of thesecommensal intra-oral bacteria. The DNA encoding the PrtR-PrtK could alsobe linked with, or inserted into, the DNA encoding extracellularproteins to produce secreted fusion proteins. Examples of extracellularproteins that could be used to produce fusion proteins with theinactivated PrtR-PrtK could be the glucosyltranferases (GTF) orfructosyltransferases (FTF). The recombinant organism would be thenre-introduced into the patients oral cavity and once colonised the oralmucosa or teeth would express the inactivated PrtR-PrtK to stimulate themucosal associated lymphoid tissue to produce neutralising antibodies.

Due to the conservation of the genes encoding PrtR-PrtK, the nucleicacid sequences of the present invention can be used in moleculardiagnostic assays for detecting P. gingivalis genetic material. Inparticular, PrtR-PrtK sequence-specific oligonucleotides can besynthesized for use as primers and/or probes in amplifying, anddetecting amplified, nucleic acids from P. gingivalis. Recent advancesin molecular biology have provided several means for enzymaticallyamplifying nucleic acid sequences. Currently the most commonly usedmethod, PCR™ (polymerase chain reaction Cetus Corporation) involved theuse of Taq Polymerase, known sequences as primers, and heating cycleswhich separate the replicating deoxyribonucleic acid (DNA) strands andexponentially amplify a gene of interest. Other amplification methodscurrently under development include LCR™ (ligase chain reaction,BioTechnica International) which utilizes DNA ligase, and a probeconsisting of two halves of a DNA segment that is complementary to thesequence of the DNA to be amplified; enzyme QB replicase (Gene-TrakSystems) and a ribonucleic acid (RNA) sequence template attached to aprobe complementary to the DNA to be copied which is used to make a DNAtemplate for exponential production of complementary RNA; and NASBA™(nucleic acid sequence-based amplification, Cangene Corporation) whichcan be performed on RNA or DNA as the nucleic acid sequence to beamplified.

Nucleic acid probes that are capable of hybridization with specific genesequences have been used successfully to detect specific pathogens inbiological specimens at levels of sensitivity approaching 10³-10⁴organisms per specimen (1990, Gene Probes for Bacteria, eds. Macario anddeMacario, Academic Press). Coupled with a method that allows foramplification of specific target DNA sequences, species-specific nucleicacid probes can greatly increase the level of sensitivity in detectingorganisms in a clinical specimen. Use of these probes may allow directdetection without relying on prior culture and/or conventionalbiochemical identification techniques. This embodiment of the presentinvention is directed to primers which amplify species-specificsequences of the genes encoding PrtR-PrtK of P. gingivalis, and toprobes which specifically hybridize with these amplified DNA fragments.By using the nucleic acid sequences of the present invention andaccording to the methods of the present invention, as few as one P.gingivalis organism may be detected in the presence of 10 ug/mlextraneous DNA.

DNA may be extracted from clinical specimens which may contain P.gingivalis using methods known in the art. For example, cells containedin the specimen may be washed in TE buffer and pelleted bycentrifugation. The cells then may be resuspended in 100 ul ofamplification reaction buffer containing detergents and proteinase K.Using the polymerase chain reaction, the resultant sample may becomposed of the cells in 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.01% gelatin, 0.45% NP40™, 0.045% Tween 20™, and 60 ug/ml proteinase K.The sample is incubated in a 55° C. water bath for 1 hour. Following theincubation, the sample is incubated at 95° C. for 10 minutes toheat-inactivate the proteinase K. The sample may then be amplified inaccordance with standard PCR protocols.

The following examples are further illustrative of the nature of thepresent invention, but it is understood that the invention is notlimited thereto. All amounts and proportions referred to herein are byweight unless otherwise indicated.

EXAMPLE 1

(1) Preparation of Antigen

A. Anion Exchange and Affinity Chromatography

P. gingivalis W50 was grown anaerobically at 37° C. on lysed horse bloodagar and in modified BM media containing 1 μg/ml hemin. Bacteria weremaintained on lysed horse blood plates by routine passage (<10 passages)and used to inoculate batch cultures. Batch culture growth in BrainHeart Infusion medium was monitored at 650 nm using a spectrophotometer(295E, Perkin-Elmer). Culture purity was checked routinely by Gramstain, microscopic examination and by using a variety of biochemicaltests. Stocks were maintained as lyophilised cultures. A culture of P.gingivalis was grown to late logarithmic phase and the cells harvestedby centrifugation (5,000×g, 20 min, 4° C.) and then resuspended in 160ml TC buffer (20 mM Tris-HCl pH 7.4 and 5 mM CaCl₂) containing 50 mMNaCl and subjected to mild sonication using a Branson Sonifier 250 withan output control of 3 and a 50% duty cycle for 15 min at 4° C. Thesonicate was centrifuged (100,000×g, 30 min, 4° C.) and the supernatantfiltered (0.22 μm) prior to anion-exchange FPLC. The sonicate wasapplied to an anion-exchange column (Hiload XK 16/10 Q Sepharose,Pharmacia-LKB) cooled to 4° C., in multiple injections using a 50 mlsuperloop (Pharmacia-LKB). The sample was eluted using a linear gradientfrom 0-100% buffer B over 90 min at a flow rate of 2.0 ml min⁻¹. Theeluant was monitored at 280 nm and collected in 6 ml fractions using aFrac 100 fraction collector (Pharmacia-LKB). Buffer A was TC buffercontaining 50 mM NaCl and buffer B was TC buffer containing 500 mM NaCl.Fractions were analysed for proteolytic and amidolytic activity usingazocasein (A-2765, Sigma Chemical Co. St Louis, Mo.),benzoyl-L-Arg-p-nitroanilide (Bz-L-Arg-pNa, Sigma) andbenzyloxycarbonyl-L-Lys-p-nitroanilide (Z-L-Lys-pNa, Calbiochem,Melbourne, Australia) vide infra. Anion-exchange fractions containingthe majority of proteolytic/amidolytic activity were pooled, washed andthen concentrated in TC buffer containing 150 mM NaCl using a centricon10 micro-concentrator (Amicon). The sample was then divided into fouraliquots and each was independently applied to a gel filtration column(Superose 12, HR 10/30, Pharmacia-LKB) using TC buffer containing 150 mMNaCl at a flow rate of 0.3 ml min⁻¹. The eluant was monitored at 280 nmand peaks collected using a Frac 100 fraction collector. The M_(r)values of eluant peaks were determined using molecular mass gelfiltration standards (Pharmacia-LKB). The peak containing the majorityof the proteolytic/amidolytic activity was concentrated using acentricon 10 micro-concentrator and then applied at a flow rate of 0.1ml min⁻¹ to an Arg-sepharose column (5 ml arginine-Sepharose 4B beads,HR 5/5 column, Pharmacia-LKB) and the unbound material collected. Thecolumn was washed with 500 mM NaCl and re-equilibrated with TC buffercontaining 50 mM NaCl. The column was first eluted with 200 mMlysine-HCl pH 7.4 in TC buffer containing 50 mM NaCl at a flow rate of0.1 ml min⁻¹. This was followed by 750 mM lysine-HCl pH 7.4 in the samebuffer. The column was then re-equilibrated with TC buffer containing 50mM NaCl and then eluted with 200 mM arginine-HCl pH 7.4 in TC buffercontaining 50 mM NaCl at a flow rate of 0.1 ml min⁻¹. The unboundmaterial collected was then re-applied to the Arg-sepharose column andthe elution steps repeated. This sequence was repeated until allproteolytic activity had bound to the column. The eluant was monitoredat 280 nm and peaks collected using a Frac 100 fraction collector. Thepeaks eluted from the Arg-sepharose by 200 mM lysine and 200 mM argininewere equilibrated with TC buffer containing 50 mM NaCl and 1.0%octyl-β-D-glucopyranoside and then applied to a Mono Q (HR 5/5)anion-exchange column and eluted using a linear gradient of 0-100%buffer B at a flow rate of 1.0 ml min⁻¹. Buffer A was TC buffercontaining 50 mM NaCl and 0.1% octyl-β-D-glucopyranoside and buffer Bwas TC buffer containing 500 mM NaCl and 0.1% octyl-β-D-glucopyranoside.The eluant was monitored at 280 nm and eluant peaks collected using aFrac 100 fraction collector.

Azocasein, Bz-L-Arg-pNa and z-L-lys-pNa were used to routinely assayFPLC fractions for proteolytic and amidolytic activity. A sample of eachfraction (20-200:1) was incubated at 37° C. with azocasein (5 mg/mlfinal concentration) in TC buffer pH 8.0 containing 150 mM NaCl and 10mM cysteine. For azocasein the reaction was stopped by the addition of30% trichloroacetic acid at 4° C. Samples were centrifuged and the A₄₄₀of the supernatant measured using a spectrophotometer (Perkin Elmer,model 552).

For the synthetic chromogenic substrates samples of each chromatographicfraction (5-50:1) were incubated at 37° C. with Bz-L-Arg-pNa orz-L-Lys-pNa (1.0 mM final concentration) in a total volume of 350:1 100mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl, 10 mM cysteine and 5mM CaCl₂. Inhibitors and activators were added to the purified enzymesin 100 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl. Absorbance wasmeasured at 410 nm in a Hewlett Packard 8452A Diode Arrayspectrophometer and the amidolytic activity expressed in U, where U=μmolsubstrate converted min⁻¹ at 37° C. Trypsin (E.C.3.4.21.4, T 8253 Sigma)was used as a standard. The protein concentration of FPLC fractions andpurified samples was determined using the Bradford protein assay(Biorad) with BSA as a standard.

A sample of the gel filtration chromatographic fraction (20 μl)exhibiting the major proteolytic and amidolytic activity was incubatedfor 4 h at 37° C. with 10 mg/ml of pure α_(s1)-casein dissolved in TCbuffer pH 8.0 containing 150 mM NaCl and 50 mM 2-mercaptoethanol.Following incubation the sample was equilibrated in 0.1% TFA (v/v)dissolved in Milli Q water (Buffer A). The sample was then applied to anHPLC reversed phase analytical column (C8, 7 μm, 4.6 mm×220 mm, AppliedBiosystems Inc. Brownlee Aquapore RP 300) and peptides eluted using alinear gradient from 0-100% buffer B over 40 min at a flow rate of 1 mlmin⁻¹ (140A solvent delivery system). Buffer B was 80% acetonitrile(v/v) in 0.1% (v/v) TFA in Milli Q water. The eluant was monitored at214 nm using a 1000S diode array detector (Applied Biosystems). Peakswere collected manually and peptides identified using a combination ofamino acid composition and sequence analyses as described previously.

SDS-PAGE was performed using a Mini protean II electrophoresis system(Biorad) with 12% (w/v), 1 mm separating gels, overlaid with 5% stackinggels (Laemmli, 1970) [Nature 277:680-685]. Two volumes of each samplewere mixed with one volume of buffer [0.5 M Tris-HCl, pH 6.8, 5% v/v2-mercaptoethanol, 10.0% w/v SDS, 0.05% w/v bromophenol blue (75% v/v)and glycerol (25% v/v)] and heated to 100° C. for 4 min unless otherwisestated. SDS-PAGE was performed at room temperature using a current of30-50 mA and a potential difference of ≦200 V. For silver staining, gelswere fixed in methanol/water/acetic acid (45/45/10, v/v/v), washed inMilli Q water, reduced with 5 μg/ml dithiothreitol and then washed inMilli Q water, all for 30 min periods. Gels were then stained for 20 minwith 0.1% w/v AgNO₃ and developed with 3% w/v sodium carbonatecontaining 0.1% v/v formaldehyde and development stopped with glacialacetic acid. For Coomassie blue staining, gels were fixed in 12% TCA andstained overnight using 0.1% (w/v) purified Coomassie brilliant blue G250 in 2% (w/v) phosphoric acid, 6% (w/v) ammonium sulphate. Gels weredestained with methanol/water/acetic acid (50/40/10, v/v/v). Proteinswere transferred onto a PVDF membrane (Problott, Applied Biosystems Inc.(ABI)) for sequence analysis using a transblot cell (Biorad). PVDFmembrane was wetted in 100% methanol and soaked in transfer buffer (10mM CAPS/10% methanol, pH 11.5). Transfer was performed using a potentialdifference of 60 V (300 mA) for 90 min. Membranes were briefly stainedusing 0.1% (w/v) Coomassie brilliant blue R 250 in methanol/water/aceticacid (5/5/1, v/v/v). Protein bands were excised, destained for 10-30 secin 50% methanol and then the N-terminal sequence determined using aHewlett Packard 10005A protein sequencer or a modified ABI 471-02Aprotein sequencer fitted with a blott cartridge.

The ultrasonication procedure was effective at releasing thecell-associated Arg- and Lys-specific proteolytic activity of P.gingivalis W50 and 15 min was required for maximal release of activity.The sonicate of P. gingivalis W50 cells contained 0.30 mg ml⁻¹ proteinand 2.6 and 2.3 μmol min⁻¹ mg protein⁻¹ activity with 1.0 mMBz-L-Arg-pNA and z-L-Lys-pNA as substrate respectively at 37° C. Thecrude sonicate was subjected to Q-sepharose anion exchange FPLC and arepresentative chromatogram is presented in FIG. 1.Proteolytic/amidolytic activity eluted as one major peak between 246-320mM NaCl (FIG. 1) which was collected, concentrated using a centricon-10(Amicon) and then applied to the Superose 12 gel filtration column (FIG.2). Molecular mass gel filtration standards were used to determine theM₄ of the peaks obtained and the major peak, which also exhibited themajor proteolytic/amidolytic activity, corresponded to 300 kDa (FIG. 2).Proteolytic/amidolytic activity was also associated with the highmolecular mass material (0.6→2.0×10⁶ Da) eluted from the gel filtrationcolumn. The 300 kDa gel filtration peak contained seven bands at 48, 45,44, 39, 27, 17 and 15 kDa on SDS-PAGE analysis (FIG. 3). The seven bandswere transblotted and subjected to N-terminal sequence analysis (Table1). This analysis revealed that the 44 kDa band contained two proteinsand the N-terminal sequences of these two 44 kDa proteins were assignedafter further purification. The N-terminal sequence of one of the 44 kDaproteins was identical to that of the 17 kDa protein and the 39 kDa and27 kDa proteins also had identical N-termini (Table 1).

TABLE 1 N-terminal sequences of proteins in the 300 kDa complexseparated by SDS-PAGE Band N-terminal sequence (kDa) 48*DVYTDHGDLYNTPVRML SEQ ID NO:1 45^(†) YTPVEEKQNGRMIVIVAKKYEGD SEQ ID NO:244^(†) SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHFL SEQ ID NO:3 44*PQSVWIERTVDLPAGTKYVAFR SEQ ID NO:4 39* ANEAKVVLAADNVWGDNTGYQFLLDA SEQ IDNO:5 27^(†) ANEAKVVLAADNVWGDNTGYQFLLDA SEQ ID NO:5 17^(†)PQSVWIERTVDLPAGTKYVAFR SEQ ID NO:4 15*^(.†) ADFTETFESSTHGEAPAEWTTIDA SEQID NO:6 *Proteins eluted from Arg-sepharose by 200 mM lysine^(†)Proteins eluted from Arg-sepharose by 200 mM arginine

Repeated gel filtration analyses of the Q-sepharose purified material orcrude sonicates indicated that the major proteolytic/amidolytic activitywas associated with a peak corresponding to 300 kDa and higher molecularmass (0.6→2×10⁶ Da) material that when boiled in SDS and subjected toSDS-PAGE analysis contained the same seven bands at 48, 45, 44, 39, 27,17 and 15 kDa (FIG. 3).

The 300 kDa gel filtration protein complex was incubated withα_(s1)-casein. The α_(s1)-casein peptides released by the action of theproteolytic activity of the 300 kDa complex were purified by RP-HPLC andidentified by amino acid composition and sequence analyses. The sites ofα_(s1)-casein cleavage by the material of the 300 kDa complex were thecarboxyl side of arginyl and lysyl residues only (FIG. 4). All arginyland lysyl residues of α_(s1)-casein were cleaved except the N-terminalArg and the Lys residues flanking the Ser(P) cluster sequence,presumably due to the high negative charge density (FIG. 4). The 300 kDacomplex was then applied to an Arg-sepharose column and washed with TCbuffer containing 500 mM NaCl (FIG. 5). The Arg-sepharose was elutedfirst with 200 mM lysine in TC buffer (FIG. 5) which eluted a smallamount of the 48 kDa, 44 kDa, 39 kDa and 15 kDa proteins of the 300 kDacomplex as shown by SDS-PAGE (FIG. 6 and Table 1). N-terminal sequenceanalysis of these transblotted proteins revealed that only one of the 44kDa proteins of the 300 kDa complex was eluted with 200 mM lysine (Table1). This fraction eluted from Arg-sepharose with 200 mM lysine containedonly Lys-specific proteolytic/amidolytic activity. Next theArg-sepharose column was eluted with 750 mM lysine (FIG. 5) whichremoved the majority of the protein bound as the undissociated 300 kDacomplex containing all seven bands (eight proteins) as shown by SDS-PAGEanalysis (FIG. 7). The 750 mM lysine eluant exhibited both Arg- andLys-specific proteolytic/amidolytic activity characteristic of the 300kDa complex. The Arg-sepharose column was then eluted with 200 mMarginine in TC buffer (FIG. 5). The 200 mM arginine eluant containedsmall amounts of the 45, 44, 27, 17 and 15 kDa proteins as shown bySDS-PAGE (FIG. 7). This fraction exhibited only Arg-specificproteolytic/amidolytic activity. N-terminal sequence analysis of thesetransblotted proteins eluted with 200 mM arginine revealed that only oneof the 44 kDa proteins of the 300 kDa complex was eluted with 200 mMarginine and this 44 kDa protein was different to the 44 kDa proteineluted with 200 mM lysine (Table 1).

The proteins eluted from the Arg-sepharose column with 200 mM lysine and200 mM arginine were washed, concentrated and equilibrated with TCbuffer containing 50 mM NaCl and 1.0% octyl-β-D-glucopyranoside andapplied independently to a Mono Q anion exchange column. Elution fromthe Mono Q column with a NaCl gradient associated the Arg-specificproteolytic activity with the 45 kDa protein with a 25 fold purificationover the original crude sonicate (Table 2, FIG. 7). The specificity ofthe 45 kDa proteinase for arginyl residues was confirmed by the enzymecleaving Bz-L-Arg-pNA but not z-L-Lys-pNA. The Arg-specific 45 kDaenzyme was activated by thiols (particularly cysteine), not inhibited byPMSF or AEBSF but inhibited by sulphydryl-directed reagents, leupeptinand EDTA (Table 3). The inhibition by EDTA could be reversed by theaddition of Ca²⁺ (Table 3). The pH optimum of the enzyme was 7.5-8.0 andactivity dropped off dramatically as the pH was lowered below 7.0. Theseresults indicate that the 45 kDa enzyme is a calcium-stabilized,Arg-specific cysteine endopeptidase. The Lys-specific activity wascharacterized using the substrate Z-L-Lys-pNA and was associated withthe 48 kDa protein purified from the 200 mM lysine eluant by Mono QFPLC. The Lys-specific enzyme was also activated by thiols and inhibitedby sulphydryl-directed reagents but was not inhibited by leupeptin orEDTA. Non-reducing SDS-PAGE without boiling of the 300 kDa complexproduced bands corresponding to the relative molecular masses ofapproximately 300, 150, 104, 88, 76 and 66 kDa.

TABLE 2 Purification of the 45 kDa Arg-specific proteinase PrtR45Proteolytic Specific Protein activity activity Purification Yield Step(mg) (U*) U mg⁻¹ fold % Sonicate 48.0 124 2.6 1 100 Anion Exchange 8.264 7.8 3 52 FPLC (Q-sepharose) Gel filtration FPLC 3.9 46 11.8 5 37(Superose 12) Affinity FPLC 0.7 17 24.3 9 14 (Arg-sepharose) Anionexchange 0.2 13 65.0 25 11 FPLC (mono Q) *Amidolytic activity using 1.0mM Bz-L-Arg-pNA; 1 unit = μmol min⁻¹ at 37° C.

TABLE 3 Effects of various activators/inhibitors on the activity of the45 kDa Arg-specific proteinase Concentration Activity Compound (mM) (%)2-mercaptoethanol 1.0 100 10.0 158 50.0 189 Dithiothreitol 1.0 109 10.0174 L-cysteine 0.1 183 1.0 320 10.0 487 PMSF^(*,†) 1.0 100 10.0 90AEBSF^(*,†) 1.0 93 10.0 80 Iodoacetic acid^(†) 1.0 82 10.0 19 PCMB^(*,†)1.0 100 10.0 14 Leupeptin^(†) 0.1 0 EDTA^(†) 1.0 100 10.0 4 50.0 0 +Ca²⁺50.0 97 o-phenanthroline^(†) 10.0 100 ^(*)PCMB, p-chloromercuribenzoicacid; PMSF, phenylmethyl sulfonyl fluoride, AEBSF,[4-(2-aminoethyl)-benzenesulfonylfluoride] ^(†)These incubations alsocontained 1.0 mM 2-mercaptoethanol

The 45, 27, 17, 15 kDa and one of the 44 kDa protein components of the300 kDa complex are encoded by the gene the PrtR as presentedschematically in FIG. 8a. The complete nucleotide sequence and deducedamino acid sequence of the PrtR is shown in FIG. 8b. Each PrtR componentis preceded by an arginyl or lysyl residue (FIGS. 8a, b) indicating thatthe polyprotein is processed by trypsin-like proteolytic specificity. Wehave designated these component parts of the 300 kDa complex, by theirrelative molecular masses as determined by SDS-PAGE, as the PrtR45,PrtR44, PrtR27, PrtR17 and PrtR15 which fit well with the predictedsizes from the deduced PrtR amino acid sequence (53.9, 44.8, 29.5, 17.5and 14.3 kDa respectively). The 44 kDa protein, the PrtR44, has beendisclosed by previous workers as a culture fluid hemagglutinin/adhesin(Pike et al., 1994)[J Biol Chem 269:406-411]. The PrtR44 has homologywith the other non-proteinase components of the multiprotein complexsuggesting a similar role for the PrtR27, PrtR17 and PrtR15 ininteracting with the protease and/or in hemagglutination or adhesion.The PrtR45 Arg-specific endopeptidase component of the PrtR complex hasthe same characteristics and N-terminal sequence as the 50 kDaArg-specific proteinase identified in the culture supernatant of P.gingivalis H66 by Chen et al. (1992)[J Biol Chem 267:18896-18901]designated Arg-gingipain.

The other proteins of the 300 kDa complex, the 48 kDa Lys-specificproteinase, the other 44 kDa protein and the 39 kDa and 15 Da proteinsare encoded by a single gene the prtK presented schematically in FIG.9a. The complete nucleotide sequence and deduced amino acid sequence ofthe PrtK is shown in FIG. 9b. The prtK is similar to the prtR in that itencodes a putative leader sequence, a prosequence followed by theproteinase domain which is then followed by sequence-related adhesinsthat have high homology with the C-terminal adhesins of the prtR. Wehave designated the 48 kDa Lys-specific proteinase the PrtK48 and itsassociated adhesins the PrtK39, PrtK15 and PrtK44 (FIGS. 9a, b) based onthe sizes measured by SDS-PAGE which fit reasonably well with thepredicted sizes from the deduced PrtK amino acid sequence (55.9, 44.8,14.3 and 47.9 kDa respectively). The PrtK48 has the same enzymecharacteristics as the 48 kDa proteinase purified from the culturesupernatant of P. gingivalis 33277 by Fujimura et al. (1993) [InfectImmun 55:716-720]. The PrtK48 also has the same N-terminal sequence andenzyme characteristics as the 60 kDa Lys-specific endopeptidasepreviously purified from the culture fluid of P. gingivalis H66 by Pikeet al. (1994) [J Biol Chem 269:406-411] and designated Lys-gingipain.The PrtK39, PrtK15 and PrtK44 are all sequence-related and have highhomology with the PrtR hemagglutinins/adhesins particularly the 15 kDaprotein which is identical in both gene products suggesting that theseproteins also are hemagglutinin/adhesins.

As the 300 kDa proteinase-adhesin complex and higher molecular massforms are composed of proteins from the two genes, the prtR and prtK, wesuggest that they be designated PrtR-PrtK complexes. The deducedmolecular mass of the mature PrtR is 160 kDa (FIGS. 9a, b) and maturePrtK is 163 kDa (FIG. 9b) such that the mass of the PrtR-PrtKheterodimer would be 323 kDa which is in good agreement with the M_(r)determined by gel filtration and non-boiling SDS-PAGE. SDS-PAGE of thesample after boiling produced the seven bands of 48, 45, 44, 39, 27, 17and 15 kDa corresponding to the domains of the two gene products, thePrtR and PrtK. These domains were only seen when the sample was boiled,with or without reducing agent, suggesting that the domains remaintightly non-covalently associated after proteolytic processing. The cellsonicate and the chromatographic fractions had minimal or no proteolyticactivity in the absence of reducing agents thus ensuring minimal enzymicactivity during the chromatographic purifications. The characterizationof the 300 kDa cell-associated complex as being composed of processeddomains of the two genes the prtR and prtK suggests that the secreted,mature PrtR and PrtK proteins associate and then are processed, perhapsautolytically. The identification of several of the domains of the PrtRand PrtK in the culture supernatant by independent groups is consistentwith the proteolytic (autolytic) processing of these polyproteins.

The relative molecular mass of the processed PrtR-PrtK complex is likelyto be attributable to the composition of 1 PrtK48+1 PrtR45+1 PrtR44+1PrtK39+1 PrtK44+1 PrtR27+1 PrtR17+1 PrtK15+1 PrtR15=294-323 kDadepending on C-terminal truncation, that is the 300 kDa complex wouldcontain the five domains of the prtR and the four domains of the prtKgene products (FIGS. 8 and 9). As high M_(r) material (0.6→2×10⁶ Da) ongel filtration (FIG. 2) was also composed of the seven PrtR-PrtK bandsthen this suggests that the 300 kDa PrtR-PrtK complexes may furtherassociate to form larger cell-associated aggregates. The high amino acidsequence homology between the PrtR44, PrtK39, PrtK44, PrtR27, PrtR17 andthe 15 kDa protein of both the PrtR and PrtK suggests that theseadhesins are responsible for the non-covalent cohesive interactionsbetween the components of the PrtR-PrtK complexes and between thecomplexes themselves in the larger aggregates. It is interesting to notethat some dissociation of the 300 kDa PrtR-PrtK complex occurred duringthe affinity chromatography on Arg-sepharose, although the majority ofthe protein eluted as the undissociated complex with 750 mM lysine. Thepartial dissociation of the complex on binding to substrate may be amechanism by which the complex targets specific host macromolecules andcells releasing the proteinase/adhesin domains at the target site onbinding.

This example describes the purification of a novel cell associatedcomplex of Arg-specific and Lys-specific proteinases andsequence-related adhesins encoded by the two genes, the prtR and prtK.

B. Ultrafiltration and Diafiltration

P. gingivalis W50 was grown anaerobically at 37° C. on lysed horse bloodagar and in modified BM media containing 1 μ/ml hemin. Bacteria weremaintained on lysed horse blood plates by routine passage (<10 passages)and used to inoculate batch cultures. Batch culture growth in BrainHeart Infusion medium was monitored at 650 nm using a spectrophotometer(295E, Perkin-Elmer). Culture purity was checked routinely by Gramstain, microscopic examination and by using a variety of biochemicaltests. Stocks were maintained as lyophilised cultures. A culture of P.gingivalis was grown to late logarithmic phase and the cells harvestedby centrifugation (5,000×g, 20 min, 4° C.). Chloroform was added to thecell pellet and after gentle mixing the suspension was left for 15 minat room temperature. Following chloroform treatment, 20 mM Tris-HCl pH8.0 buffer containing 50 mM NaCl was added and gently mixed. Thismixture was then centrifuged (100,000×g, 30 min, 4° C.) and thesupernatant diafiltered through a 100,000 M_(r) cut-off membrane(Amicon) with five volumes of distilled water. This purifies andinactivates by oxidation the 294≅323 kDa PrtR-PrtK which is freeze driedand used as an immunogen. The PrtR-PrtK purified by diafiltration wascomposed of 48, 45, 44, 39, 27 17 and 15 kDa components as shown bySDS-PAGE (FIG. 10).

(2) Preparation of Antibodies

Polyclonal antiserum to PrtR-PrtK was raised in a rabbit by immunizingwith the O₂-inactivated PrtR-PrtK subcutaneously. The rabbit wasimmunized at day 0 with 40 μg of protein in incomplete Freund'sadjuvant, day 14 with 90 μg of protein in incomplete Freund's adjuvant,and day 28 with 60 μg of protein in incomplete Freund's adjuvant.Immunizations were carried out using standard procedures. Polyclonalantisera having a high titre against P. gingivalis was obtained. Ifdesired the antibodies directed specifically against P. gingivalis canbe obtained using standard procedures.

EXAMPLE 2

Methods and compounds for vaccine formulations related to PrtR-PrtK.

This embodiment of the present invention is to provide PrtR-PrtK proteinto be used in as an immunogen in a prophylactic and/or therapeuticvaccine for active immunization to protect against or treat infectionscaused by P. gingivalis. For vaccine purposes, an antigen of P.gingivalis comprising a bacterial protein should be immunogenic, andinduce functional antibodies directed to one or more surface-exposedepitopes on intact bacteria, wherein the epitope(s) are conservedamongst strains of P. gingivalis.

In one illustration of the PrtR-PrtK protein having the propertiesdesirable of a vaccine antigen, the protein was purified from P.gingivalis using the method described herein in Example 1. Mice wereimmunized with the purified inactivated PrtR-PrtK protein (25 ug) withadjuvant (20 ug of QS21) two times at four week intervals. The purifiedPrtR-PrtK was inactivated by air oxidation. Blood from the immunizedmice was drawn 32 days after the last immunization and the immune serawas pooled. The pooled immune sera was assayed against whole bacteria(P. gingivalis strain W50) by an enzyme linked immunosorbent assay(ELISA). For the whole cell ELISA, overnight cultures of bacteria wereharvested by a swab and suspended in PBS to an absorbance of 0.1 at 600nm. Aliquots (100 ul) of the bacterial suspension were added to thewells of a 96 well microtiter plate and dried overnight at roomtemperature. The plates were blocked with 100 ul of 0.1% (w/v) gelatinin PBS. This, and all remaining incubations, were for one hour at roomtemperature unless otherwise specified. The blocking solution wasremoved and 100 ul of the immune sera, diluted in PBS with 0.1% (w/v)gelatin, was added to the wells and incubated. After washing three timeswith PBS, the bound antibodies were detected by incubating with 100 ulof alkaline phosphatase conjugated recombinant protein G (1:1500 in PBSwith 0.1% (w/v) gelatin). The plates were washed and colour developmentwas facilitated by the addition of 100 ul/well of p-nitrophenylphosphate (2 mg/ml in diethanolamine). After 30 minutes, the reactionwas stopped by adding 50 ul of 3M NaOH. The absorbance was read at 492nm using an ELISA reader. Endpoint titers were determined as thereciprocal of the dilution at which the absorbance was greater than thatof the blank wells. The results demonstrated that immunization withinactivated PrtR-PrtK elicit antibodies which can bind to one or moresurface-exposed epitopes on intact P. gingivalis.

Additional evidence supporting the immunogenicity of the PrtR-PrtKprotein comes from a study of the human immune response to the PrtR-PrtKof P. gingivalis in which 86% of 43 patients with adult periodontitishad specific IgG in their sera to the PrtR-PrtK.

Another illustration of a desirable vaccine antigen is theO₂-inactivated PrtR-PrtK. It has been demonstrated that the cell surfacePrtR-PrtK is the target of bactericidal antibody generated fromimmunization with the inactivated protein. Polyclonal antiserum toPrtR-PrtK was raised in a rabbit by immunizing with the inactivatedPrtR-PrtK subcutaneously. A rabbit was immunized at day 0 with 40 μg ofprotein in incomplete Freund's adjuvant, day 14 with 90 μg of protein inincomplete Freund's adjuvant, and day 28 with 60 μg of protein inincomplete Freund's adjuvant. The resultant antiserum was tested for itsbactericidal activity against strain W50 of P. gingivalis. The bacteriawere grown to logarithmic phase in brain-heart infusion (BHI) broth. Analiquot of the bacterial culture was diluted to 5×10⁴ colony formingunits (CFU) per ml in 10% bovine serum albumin in a balanced saltsolution. The bactericidal assay reaction contained bacteria, polyclonalantiserum to inactivated PrtR-PrtK protein, a complement sourceconsisting of normal human serum which was absorbed with protein G toremove antibodies, and the balanced salt solution. All reagents wereadded to the reaction to yield a 250 μl volume. Aliquots of 25 μl of thereaction were removed and plated in triplicate on BHI agar at times 0and 60 minutes. The plates were incubated and colonies were counted thenext day. The percent killing was calculated using the average of thethree triplicate values at the 2 times. A representative example of datagenerated by the bactericidal assays is shown in Table 4. The resultsindicate that the polyclonal antiserum raised to the inactivatedPrtR-PrtK is bactericidal for P. gingivalis. As illustrated by Table 4,controls show that the antiserum does not kill bacteria in the absenceof complement, and that the complement source does not kill the bacteriain the absence of the antiserum, indicating that the bactericidalactivity is antibody directed and complement mediated.

TABLE 4 Bactericidal activity of anti-(PrtR-PrtK) antibody CFU at CFU atPercent Sample Antiserum Complement time 0 time 60 killing 1 10 μl 22 μl225 0 100% 2 10 μl  0 227 390 0% 3  0 22 μl 254 286 0%

In further illustrating that the PrtR-PrtK protein possesses propertiesdesirable of a vaccine antigen, pooled immune sera raised to strain W50was shown to have cross-reactivity with heterologous strains. The pooledimmune sera, prepared against PrtR-PrtK protein as described above, wasexamined for cross-reactivity with nine P. gingivalis strains fromdiverse clinical and geographical sources. Bacteria from each culturewere harvested by swabs and suspended in PBS to an optical absorbance of1.0 at 600nm. A microliter of each suspension was applied to anitrocellulose membrane and allowed to dry. The membrane was incubatedone hour at room temperature in a solution of 5% non-fat dry milk in PBSto block the residual binding sites of the membrane. The membrane waswashed twice with PBS, and then immersed in the blocking solutioncontaining the immune sera diluted to 1:1000. The membrane was incubatedwith the antibody overnight at 46° C. with gentle shaking. The membranewas washed three times with PBS and then incubated for 2 hours at roomtemperature with alkaline phosphatase conjugated recombinant protein G(1:1500 in PBS with 5% non-fat dry milk). The membrane was washed threetimes with PBS and bound antibody was detected by the addition ofsubstrate. The immune sera reacted with all strains as strongly, or to agreater extent than, strain W50. Thus, the antibodies elicited byimmunization of the PrtR-PrtK protein isolated from strain W50cross-reacted with all heterologous strains tested.

For vaccine development, PrtR-PrtK may be purified from a hostcontaining a recombinant vector which expresses PrtR-PrtK. Such hostsinclude, but are not limited to, bacterial transformants, yeasttransformants, filamentous fungal transformants, and cultured cells thathave been either infected or transfected with a vector which encodesPrtR-PrtK. Many methods are known for the introduction of a vaccineformulation into the human or animal to be vaccinated. These include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, ocular, intranasal, and oral administration.The vaccine may further comprise a physiological carrier such as asolution, a polymer or liposomes; and an adjuvant, or a combinationthereof.

EXAMPLE 3

Protective Efficacy of Immunisation with the PrtR-PrtK Complex in anAnimal Model

Various preparations of purified P. gingivalis proteins were tested inthe mouse abscess model. This model is loosely based on the methodsdescribed by Kesavalu et al (1992) [Infect Immun 60:1455-1464]. Atypical experiment is outlined below. Briefly BALB/c mice were obtainedfrom ARC (Perth, Australia) and were immunised subcutaneously in thescruff of the neck with the preparations and doses according to Table 5before challenge with live P. gingivalis strain W50, which was given at10 weeks of age. Mice were given 2 doses of vaccine at 4 and 1 weeksbefore challenge. Formalin killed P. gingivalis W50 cells were preparedby incubating an aliquot of cells in 0.5% (vol/vol) of buffered formalsaline overnight at 4° C. The chloroform extract of P. gingivalis wasprepared as detailed in Example 2. Purification of PrtR-PrtK complex wasperformed as detailed in Example 1. The PrtR-PrtK domains were preparedby taking the PrtR-PrtK complex and incubating in the presence of 50 mM2-mercaptoethanol for 8 h at 4° C. This resulted in the breakdown of thePrtR-PrtK complex to domains that were 15-115 kDa proteins as shown bygel filtration FPLC and SDS-PAGE as performed in Example 1.

All preparations were emulsified with an equal volume of Freund'sIncomplete Adjuvant (FIA; Sigma) prior to injection.

Animals were bled before and 1 week after the immunisation schedule.Sera were screened by ELISA using a P. gingivalis sonicate (prepared asin Example 1) as the adsorbed antigen. The immunogenicity of thepurified PrtR-PrtK complex is shown in FIG. 11.

TABLE 5 Immunization schedule No. of Group Doses Treatment n 1 2 1 × 10⁹Formalin 11 killed P. gingivalis cells in FIA¹ 2 2 Chloroform extracted10 P. gingivalis proteins in FIA 3 2 Affinity purified P. gingivalis 5PrtR-PrtK complex in FIA 4 2 PrtR-PrtK Domains in FIA 10 5 2Tris-cysteine buffer in FIA 10 6 2 Tris-cysteine buffer 10 ¹FIA =Freunds incomplete adjuvant

For the preparation of the bacterial challenge P. gingivalis cells weregrown at 37° C. on lysed horse blood agar (HBA) plates until day 3 or 4in an anaerobic chamber (Mark 3 Anaerobic Workstation, Don WhitleyScientific Limited; with an air mixture of 8% H₂, 12% CO₂, 80% N₂), thenpassaged into 20 ml of brain heart infusion broth (BHIB; Oxoid)supplemented with 0.5 g/L cysteine and 1 mg/L haemin for 24 hours in astandard incubator at 37° C. Finally, 3 ml of this culture was added to400 ml of BHIB-cysteine media and incubated for approximately 15 hoursin a standard incubator at 37° C., until the optical density at 650 nmreached 0.18. The cells were then pelleted by centrifugation at 10,000 gfor 30 minutes using a JA10 rotor in a Beckman High Speed centrifuge andthen resuspended to a final dilution of 3×10¹⁰ cells per ml inBHIB-cysteine media according to previously established growth curvesfor the W50 strain used in these experiments. Mice were marked foridentification, their backs and chests shaved to make measurement oflesions possible, then weighed prior to inoculation with the challengedose at a single site in the middle of the back. A 0.1 ml dose was givenrepresenting a predicted challenge dose of 3×10⁹ bacteria per mouse. Theinoculum dose was confirmed by culturing various dilutions of thechallenge dose on lysed HBA plates and examining the number of colonies7 days later.

Following challenge mice were examined daily for the number and size oflesions on their body and their size estimated by measuring theapproximate surface area in mm² involved. Previous experiments had shownthat in unimmunized mice, lesions developed on the belly of the micefollowing inoculation of live bacteria into the back or side. Anydistressed animals were culled. Observations were carried out over twoweeks and a summary of one such experiment is summarised below in Table6. In this experiment while a dose of 3×10⁹ bacteria per mouse was thedesired number of bacteria, after plating out of the inoculum it wascalculated that each mouse actually received a challenge dose of3.17×10⁹ live P. gingivalis bacteria strain W50.

When mice were immunised with the various P. gingivalis fractionssignificant reductions (p<0.05) were seen in the size of the lesionswith whole formalin killed P. gingivalis strain W50 cells (Group 1), thechloroform extracted proteins (Group 2) and the PrtR-PrtK complex (Group3) when compared with the lesion size of the animals receiving FIA(Group 5) (Table 6). The PrtR-PrtK domains (Group 4) of the broken downPrtR-PrtK complex did not significantly reduce lesion size compared withthe control (Group 5). These results clearly show that the complex workseffectively as an immunogen whereas the PrtR-PrtK domains (15-115 kDaproteins) do not. The only group of animals that had a number of animals(40%) that exhibited no visible lesions at all was the PrtR-PrtK complexgroup (Group 3). All other groups, including formalin killed cells(Group 1), had all animals exhibiting visible lesions indicating thatthe PrtR-PrtK complex was a better immunogen than formalin killed cells.

TABLE 6 Immunisation with the PrtR-PrtK complex can protect mice fromchallenge with P. gingivalis Lesion size Group Mean maximum lesion sizemm² p* 1  30.2 ± 28.4^(†) 0.0008 2 39.0 ± 33.2 0.009 3 30.0 ± 36.00.0028 4 88.3 ± 32.2 NS 5 86.8 ± 41.1 — 6 201.7 ± 125.8 0.012*probability calculated by Mann Whitney rank sum test comparing Group 5with other groups ^(†)mean ± SD

EXAMPLE 4

Cloning and Sequence Analysis of the prtR and prtK Genes

Bacterial Strains

P. gingivalis W50 was grown in modified BM medium supplemented with 1μg/ml haemin in an atmosphere of 10% CO₂, 10% H₂ and 80% N₂ at 37° C.Escherichia coli JM109 and Escherichia coli LE392 were grown in LBmedium at 37° C. Escherichia coli strains harbouring pUC18 plasmids weregrown in LB medium supplemented with 100 μg/ml ampicillin at 37° C.

Genomic Library Construction

Chromosomal DNA was isolated from P. gingivalis W50 as described bySmith et al, [Oral Microbiol. Immunol. 4:47-51 (1989)] except that cellswere pelleted from a 500 ml late-exponential culture. The genomiclibrary was constructed from BamHI partially-digested W50 DNA which waspartially-filled with dGTP and dATP and ligated into LambdaGEM®-12 XhoIhalf-site arms (Promega) and packaged using Packagene® (Promega).

prtR gene characterisation: The genomic library was screened usingdegenerate synthetic oligonucleotides derived from the N-terminalsequence information of the purified PrtR45. The oligonucleotide probeswere based on the amino acid sequence YEGDIKD (antisense) and KDFVDWKNQ(sense) and were 5′end-labelled using γ³²P ATP and T4 polynucleotidekinase. Approximately 1.5×10⁴ phage were screened by lifting onto Nylonmembrane filters and hybridised with radiolabeled oligonucleotidesovernight in hybridisation buffer: 6×SSC (SSC is 15 mM sodium citrate,150 mM NaCl pH 8.0), 0.25% SDS, 5×Denhardt's solution and 100 μg/mlsalmon sperm DNA at 44° C. Filters were washed extensively in a solutionof 5×SSC containing 0.01% SDS (w/v) at 44° C. Positively-hybridisingplaques were purified. Standard protocols for end-labelling ofoligonucleotides and screening procedures were essentially as describedin Sambrook et al. (1989) [Molecular Cloning: A Laboratory Manual; 2nded., Cold Spring Harbour Laboratory Press]. Lambda clone four with aninsert size of approximately 15 kb was selected and this fragmentcontained the entire prtR gene. The 15 kb fragment was cut withappropriate restriction enzymes and the fragments generated subclonedinto pUC18. Escherichia coli JM109 was transformed with the recombinantplasmids using electroporation.

prtk gene characterisation: The 5′ portion of the gene encoding PrtK wasisolated from the same genomic library described above. The genomiclibrary was screened using a degenerate synthetic oligonucleotidederived from the N-terminal sequence information of the purified PrtK48.The oligonucleotide probes were sense to the amino acid sequenceDVYTDHGD and radiolabelled as described above. Hybridisation and washingconditions were as described above except that the temperature was 48°C. and the filters were washed extensively in a solution of 3×SSCcontaining 0.01% SDS (w/v) at 48° C. Lambda clone 12 with an insert sizeof approximately 15 kb was selected and digested with BamHI and a 3.3 kbfragment was ligated into plasmid BamHI-BAP pUC18 and Escherichia coliJM109 transformed with the recombinant plasmid as described previously.Due to an internal BamHI site within prtK, the 3.3 kb BamHI fragmentcontained the 5′ portion of prtK which constituted the end of the lambda12 clone. Sequence characterisation of the 3.3 kb BamHI fragment showedthat the DNA sequence encoding PrtK48 contains an internal EcoRI site.Subsequently, a second oligonucleotide probe (lysir) specific to thesequence THIGAH which is found within the PrtK48 was generated todetermine a suitable strategy for cloning the 3′ end of prtK Southernblot analysis of genomic DNA indicated that a 7.5 kb EcoRI fragmentcontained the entire 3′ portion of prtK. In order to characterise the 3′end of the prtK gene a second genomic library was prepared. EcoRIdigested DNA fragments of 6-8 kb were purified from an agarose gel andsubsequently ligated to EcoRI digested Lambda Zap II-calf intestinalphosphatase-treated vector (Stratagene). The genomic library enrichedfor 6-8 kb P. gingivalis EcoRI fragments was packaged using GigapackIIIGold packaging extract (Stratagene) according to the manufacturer'sinstructions. The library was screened as described previously, usingoligonucleotide lysur except that hybridisation temperatures were 42° C.and filters were washed to 3×SSC containing 0.01% SDS (w/v) at 42° C. Invivo excision of the Lambda Zap II positive genomic clone was performed(Stratagene instruction manual) to excise the pBluescript phagemid whichwas subsequently sequenced to generate the sequence informationcorresponding to the 3′ end of the prtK gene.

DNA Sequencing. Double-stranded plasmid template DNA prepared followingthe procedure of Li and Schweizer [Focus 15:19-20 (1993)] was sequencedin both directions using DNA sequence-derived, syntheticoligonucleotides, following the di-deoxy termination method [Proc. Natl.Acad. Sci. U.S.A. 74:5463-5467 (1977)], using the Sequenase version 2.0nucleotide sequencing kit purchased from United States Biochemicals.Nucleotide and protein sequence data were analysed using programmesuites accessed by the Australian National Genomic Information Service(ANGIS).

EXAMPLE 5

The following is an example of a proposed toothpaste formulationcontaining anti-(PrtR-PrtK) antibodies.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Glycerol 20.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Chlorhexidine gluconate0.01 Dextranase 0.01 Goat serum containing anti-(PrtR-PrtK) 0.2 Waterbalance

EXAMPLE 6

The following is an example of a proposed toothpaste formulation

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase0.01 Bovine serum containing anti-(PtR-PrtK) 0.2 Water balance

EXAMPLE 7

The following is an example of a proposed toothpaste formulation.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Bovine milk Ig containing anti-(PrtR-PrtK) 0.1 Water balance

EXAMPLE 8

The following is an example of a proposed toothpaste formulation.

Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium Monofluorophosphate 0.76Sodium saccharine 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavouroil 0.95 anti-(PrtR-PrtK) mouse monoclonal 0.3 sodium lauryl sulphate2.00

EXAMPLE 9

The following is an example of a proposed liquid toothpaste formulation.

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3Chlorhexidine gluconate 0.01 Ethanol 3.0 Equine Ig containinganti-(PrtR-PrtK) 0.2 Linolic acid 0.05 Water balance

EXAMPLE 10

The following is an example of a proposed mouthwash formulation.

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.3 Rabbit Ig containing anti-(PrtR-PrtK) 0.2 Waterbalance

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

15 1 17 PRT Organism Porphyromonas gingivalis 1 Asp Val Tyr Thr Asp HisGly Asp Leu Tyr Asn Thr Pro Val Arg Met 1 5 10 15 Leu 2 23 PRTPorphyromonas gingivalis 2 Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly ArgMet Ile Val Ile Val 1 5 10 15 Ala Lys Lys Tyr Glu Gly Asp 20 3 42 PRTPorphyromonas gingivalis 3 Ser Gly Gln Ala Glu Ile Val Leu Glu Ala HisAsp Val Trp Asn Asp 1 5 10 15 Gly Ser Gly Tyr Gln Ile Leu Leu Asp AlaAsp His Asp Gln Tyr Gly 20 25 30 Gln Val Ile Pro Ser Asp Thr His Phe Leu35 40 4 22 PRT Porphyromonas gingivalis 4 Pro Gln Ser Val Trp Ile GluArg Thr Val Asp Leu Pro Ala Gly Thr 1 5 10 15 Lys Tyr Val Ala Phe Arg 205 26 PRT Porphyromonas gingivalis 5 Ala Asn Glu Ala Lys Val Val Leu AlaAla Asp Asn Val Trp Gly Asp 1 5 10 15 Asn Thr Gly Tyr Gln Phe Leu LeuAsp Ala 20 25 6 24 PRT Porphyromonas gingivalis 6 Ala Asp Phe Thr GluThr Phe Glu Ser Ser Thr His Gly Glu Ala Pro 1 5 10 15 Ala Glu Trp ThrThr Ile Asp Ala 20 7 5280 DNA Porphyromonas gingivalis 7 gaattttgtctcccaagaag actttataat gcataaatac agaaggggta ctacacagta 60 aaatcatattctaatttcat caaaatgaaa aacttgaaca agtttgtttc gattgctctt 120 tgctcttccttattaggagg aatggcattt gcgcagcaga cagagttggg acgcaatccg 180 aatgtgagattgctcgaatc cactcagcaa tcggtgacaa aggttcagtt ccgtatggac 240 aacctcaagttcaccgaagt tcaaacccct aagggaatcg gacaagtgcc gacctataca 300 gaaggggttaatctttctga aaaagggatg cctacgcttc ccattctatc acgctctttg 360 gcggtttcagacactcgtga gatgaaggta gaggttgttt cctcaaagtt catcgaaaag 420 aaaaatgtcctgattgcacc ctccaagggc atgattatgc gtaacgaaga tccgaaaaag 480 atcccttacgtttatggaaa gacgtactcg caaaacaaat tcttcccggg agagatcgcc 540 acgcttgatgatccttttat ccttcgtgat gtgcgtggac aggttgtaaa ctttgcgcct 600 ttgcagtataaccctgtgac aaagacgttg cgcatctata cggaaatcac tgtggcagtg 660 agcgaaacttcggaacaagg caaaaatatt ctgaacaaga aaggtacatt tgccggcttt 720 gaagacacatacaagcgcat gttcatgaac tacgagccag ggcgttacac accggtagag 780 gaaaaacaaaatggtcgtat gatcgtcatc gtagccaaaa agtatgaggg agatattaaa 840 gatttcgttgattggaaaaa ccaacgcggt ctccgtaccg aggtgaaagt ggcagaagat 900 attgcttctcccgttacagc taatgctatt cagcaattcg ttaagcaaga atacgagaaa 960 gaaggtaatgatttgaccta tgttcttttg attggcgatc acaaagatat tcctgccaaa 1020 attactccggggatcaaatc cgaccaggta tatggacaaa tagtaggtaa tgaccactac 1080 aacgaagtcttcatcggtcg tttctcatgt gagagcaaag aggatctgaa gacacaaatc 1140 gatcggactattcactatga gcgcaatata accacggaag acaaatggct cggtcaggct 1200 ctttgtattgcttcggctga aggaggccca tccgcagaca atggtgaaag tgatatccag 1260 catgagaatgtaatcgccaa tctgcttacc cagtatggtt ataccaagat tatcaaatgt 1320 tatgatccgggagtaactcc taaaaacatt attgatgctt tcaacggagg aatctcgttg 1380 gccaactatacgggccacgg tagcgaaaca gcttggggta cgtctcactt cggcaccact 1440 catgtgaagcagcttaccaa cagcaaccag ctaccgttta ttttcgacgt agcttgtgtg 1500 aatggcgatttcctattcag catgccttgt ttcgcagaag cattgatgcg tgcacaaaaa 1560 gatggtaagccgacaggtac tgttgctatc atagcgtcta cgatcaacca gtcttgggct 1620 tctcctatgcgcgggcagga tgagatgaac gaaattctgt gcgaaaaaca cccgaacaac 1680 atcaagcgtactttcggtgg tgtcaccatg aacggtatgt ttgctatggt ggaaaagtat 1740 aaaaaggatggtgagaagat gctcgacaca tggactgtat tcggcgaccc ctcgctgctc 1800 gttcgtacacttgtcccgac caaaatgcag gttacggctc cggctcagat taatttgacg 1860 gatgcttcagtcaacgtatc ttgcgattat aatggtgcta ttgctaccat ttcagccaat 1920 ggaaagatgttcggttctgc agttgtcgaa aatggaacag ctacaatcaa tctgacaggt 1980 ctgacaaatgaaagcacgct tacccttaca gtagttggtt acaacaaaga gacggttatt 2040 aagaccatcaacactaatgg tgagcctaac ccctaccagc ctgtttccaa cttgactgct 2100 acaacgcagggtcagaaagt aacgctcaag tgggatgcac cgagcacgaa aaccaatgca 2160 accactaataccgctcgcag cgtggatggc atacgagaac tggttcttct gtcagtcagc 2220 gatgcccccgaacttcttcg cagcggtcag gccgagattg ttcttgaagc tcacgatgtt 2280 tggaatgatggatccggtta tcagattctt ttggatgcag accatgatca atatggacag 2340 gttatacccagtgataccca tactctttgg ccgaactgta gtgtcccggc caatctgttc 2400 gctccgttcgaatatactgt tccggaaaat gcagatcctt cttgttcccc taccaatatg 2460 ataatggatggtactgcatc cgttaatata ccggccggaa cttatgactt tgcaattgct 2520 gctcctcaagcaaatgcaaa gatttggatt gccggacaag gaccgacgaa agaagatgat 2580 tatgtatttgaagccggtaa aaaataccat ttccttatga agaagatggg tagcggtgat 2640 ggaactgaattgactataag cgaaggtggt ggaagcgatt acacctatac tgtctatcgt 2700 gacggcacgaagatcaagga aggtctgacg gctacgacat tcgaagaaga cggtgtagct 2760 acgggcaatcatgagtattg cgtggaagtt aagtacacag ccggcgtatc tccgaaggta 2820 tgtaaagacgttacggtaga aggatccaat gaatttgctc ctgtacagaa cctgaccggt 2880 agtgcagtcggccagaaagt aacgcttaag tgggatgcac ctaatggtac cccgaatcca 2940 aatccaaatccgaatccaaa tccgaatccc ggaacaacta cactttccga atcattcgaa 3000 aatggtattcctgcctcatg gaagacgatc gatgcagacg gtgacgggca tggctggaag 3060 cctggaaatgctcccggaat cgctggctac aatagcaatg gttgtgtata ttcagagtca 3120 ttcggtcttggtggtatagg agttcttacc cctgacaact atctgataac accggcattg 3180 gatttgcctaacggaggtaa gttgactttc tgggtatgcg cacaggatgc taattatgca 3240 tccgagcactatgcggtgta tgcatcttcg accggtaacg atgcatccaa cttcacgaat 3300 gctttgttggaagagacgat tacggcaaaa ggtgttcgct cgccggaagc tatgcgtggt 3360 cgtatacagggtacttggcg ccagaagacg gtagaccttc ccgcaggtac gaaatatgtt 3420 gctttccgtcacttccaaag caccgatatg ttctacatcg accttgatga ggttgagatc 3480 aaggccaatggcaagcgcgc agacttcacg gaaacgttcg agtcttctac tcatggagag 3540 gcaccagcggaatggactac tatcgatgcc gatggcgatg gtcagggttg gctctgtctg 3600 tcttccggacaattggactg gctgacagct catggcggca ccaacgtagt aagctctttc 3660 tcatggaatggaatggcttt gaatcctgat aactatctca tctcaaagga tgttacaggc 3720 gcaacgaaggtaaagtacta ctatgcagtc aacgacggtt ttcccgggga tcactatgcg 3780 gtgatgatctccaagacggg cacgaacgcc ggagacttca cggttgtttt cgaagaaacg 3840 cctaacggaataaataaggg cggagcaaga ttcggtcttt ccacggaagc cgatggcgcc 3900 aaacctcaaagtgtatggat cgagcgtacg gtagatttgc ctgcgggcac gaagtatgtt 3960 gctttccgtcactacaattg ctcggatttg aactacattc ttttggatga tattcagttc 4020 accatgggtggcagccccac cccgaccgat tatacctaca cggtgtatcg tgatggtacg 4080 aagatcaaggaaggtttgac cgaaacgacc ttcgaagaag acggcgtagc tacgggcaat 4140 catgagtattgcgtggaagt gaagtacaca gccggcgtat ctccgaagaa atgtgtaaac 4200 gtaactgttaattcgacaca gttcaatcct gtaaagaacc tgaaggcaca accggatggc 4260 ggcgacgtggttctcaagtg ggaagccccg agcgcaaaga agacagaagg ttctcgtgaa 4320 gtaaaacggatcggagacgg tcttttcgtt acgatcgaac ctgcaaacga tgtacgtgcc 4380 aacgaagccaaggttgtgct cgcagcagac aacgtatggg gagacaatac gggttaccag 4440 ttcttgttggatgccgatca caatacattc ggaagtgtca ttccggcaac cggtcctctc 4500 tttaccggaacagcttcttc cgatctttac agtgcgaact tcgagtcttt gatcccggcc 4560 aatgccgatcctgttgttac tacacagaat attatcgtta caggacaggg tgaagttgta 4620 atccccggtggtgtttacga ctattgcatt acgaacccgg aacctgcatc cggaaagatg 4680 tggatcgcaggagatggagg caaccagcct gcacgttatg acgatttcac attcgaagca 4740 ggcaagaagtacaccttcac gatgcgtcgc gccggaatgg gagatggaac tgatatggaa 4800 gtcgaagacgattcacctgc aagctatacc tatacagtct atcgtgacgg cacgaagatc 4860 aaggaaggtctgaccgaaac gacctaccgc gatgcaggaa tgagtgcaca atctcatgag 4920 tattgcgtggaagttaagta cacagccggc gtatctccga aggtttgtgt ggattatatt 4980 cctgacggagtggcagacgt aacggctcag aagccttaca cgctgacagt tgtaggaaag 5040 acgatcacggtaacttgcca aggcgaagct atgatctacg acatgaacgg tcgtcgtctg 5100 gcagccggtcgcaacacggt tgtttacacg gctcagggcg gctactatgc agttatggtt 5160 gtcgttgacggcaagtctta cgtagagaaa ctcgctatca agtaaatctg tcttggactc 5220 ggagactttgtgcagacact tttaagatag gtctgtaatt gtctcagagt atgaatcggt 5280 8 6000 DNAPorphyromonas gingivalis 8 ggatcctacg cccgataccc atactcgaag cctttgctcagtaccatcct gcagaaggtt 60 actctttcgc atatagtgac cctcttttct ctcagcataatggtacctat catatcagta 120 aggggcgtat tgtcttttcg aacaatgtac agcccgagaactctttactt ccacatcaca 180 cccccgactc cttagtcaag gatctttttt cccctttcccctccgctctc ttcctcatgc 240 tggactgact taaccttggt ctgctctact tttcggttgtaaatacatgc aacacaataa 300 ctttaattgt tgttagacaa cacttttaca agactctgacttttaatgag gtggagcatg 360 aaccttttcc tctttcatct tctccttcag attacagtcaatattttggc aaaaggctaa 420 ttgacagcct tttataaggg ttaatccctt gtcgcttatattgaaaacat gttctttata 480 atccgatact cttcttaaat cgaatttttt ctctaaattgcgccgcaaca aaactccttg 540 agaaaagtac caatagaaat agaaggtagc attttgcctttaaattcctt ttcttttctt 600 ggattgttct tgaaatgaat cttatttgtg gattttttttgtttttttaa cccggccgtg 660 gttctctgaa tcacgaccat aaattgtttt aaagtatgaggaaattatta ttgctgatcg 720 cggcgtccct tttgggagtt ggtctttacg cccaaagcgccaagattaag cttgatgctc 780 cgactactcg aacgacatgt acgaacaata gcttcaagcagttcgatgca agcttttcgt 840 tcaatgaagt cgagctgaca aaggtggaga ccaaaggtggtactttcgcc tcagtgtcaa 900 ttccgggtgc attcccgacc ggtgaggttg gttctcccgaagtgccagca gttaggaagt 960 tgattgctgt gcctgtcgga gccacacctg ttgttcgcgtgaaaagtttt accgagcaag 1020 tttactctct gaaccaatac ggttccgaaa aactcatgccacatcaaccc tctatgagca 1080 agagtgatga tcccgaaaag gttcccttcg tttacaatgctgctgcttat gcacgcaaag 1140 gttttgtcgg acaagaactg acccaagtag aaatgttggggacaatgcgt ggtgttcgca 1200 ttgcagctct taccattaat cctgttcagt atgatgtggttgcaaaccaa ttgaaggtta 1260 gaaacaacat cgaaattgaa gtaagctttc aaggagctgatgaagtagct acacaacgtt 1320 tgtatgatgc ttcttttagc ccttatttcg aaacagcttataaacagctc ttcaatagag 1380 atgtttatac agatcatggc gacttgtata atacgccggttcgtatgctt gttgttgcag 1440 gtgcaaaatt caaagaagct ctcaagcctt ggctcacttggaaggctcaa aagggcttct 1500 atctggatgt gcattacaca gacgaagctg aagtaggaacgacaaacgcc tctatcaagg 1560 catttattca caagaaatac aatgatggat tggcagctagtgctgctccg gtcttcttgg 1620 ctttggttgg tgacactgac gttattagcg gagaaaaaggaaagaaaaca aaaaaagtta 1680 ccgacttgta ttacagtgca gtcgatggcg actatttccctgaaatgtat actttccgta 1740 tgtctgcttc ttccccagaa gaactgacga acatcattgataaggtattg atgtatgaaa 1800 aggctactat gccagataag agttatttgg agaaagttctcttgattgca ggtgcagatt 1860 atagctggaa ttcccaggta ggtcagccaa ccattaaatacggtatgcag tactactaca 1920 accaagagca tggttatacc gacgtgtaca actatctcaaagccccttat acaggttgct 1980 acagtcattt gaataccgga gtcagctttg caaactatacagcgcatgga tctgagaccg 2040 catgggctga tccacttctg actacttctc aactgaaagcactcactaat aaggacaaat 2100 acttcttagc tattggcaac tgctgtatta cagctcaattcgattatgta cagccttgct 2160 tcggagaggt aataactcgc gttaaggaga aaggggcttatgcctatatc ggttcatctc 2220 caaattctta ttggggcgag gactactatt ggagtgtgggtgctaatgcc gtatttggtg 2280 ttcagcctac ttttgaaggt acgtctatgg gttcttatgatgctacattc ttggaggatt 2340 cgtacaacac agtgaattct attatgtggg caggtaatcttgccgctact catgctggaa 2400 atatcggcaa tattacccat attggtgctc attactattgggaagcttat catgtccttg 2460 gcgatggttc ggttatgcct tatcgtgcaa tgcctaagaccaatacttat acgcttcctg 2520 cctctttgcc tcagaatcag gcttcttata gcattcaggcttctgccggt tcttacgtag 2580 ctatttctaa agatggagtt ttgtatggaa caggtgttgctaatgccagc ggtgttgcga 2640 ctgtgagtat gactaagcag attacggaaa atggtaattatgatgtagtt atcactcgct 2700 ctaattatct tcctgtgatc aagcaaattc aggtaggtgagcctagcccc taccagcccg 2760 tttccaactt gacagctaca acgcagggtc agaaagtaacgctcaagtgg gaagcaccga 2820 gcgcaaagaa ggcagaaggt tcccgtgaag taaaacggatcggagacggt cttttcgtta 2880 cgatcgaacc tgcaaacgat gtacgtgcca acgaagccaaggttgtgctt gcggcagaca 2940 acgtatgggg agacaatacg ggttaccagt tcttgttggatgccgatcac aatacattcg 3000 gaagtgtcat tccggcaacc ggtcctctct ttaccggaacagcttcttcc aatctttaca 3060 gtgcgaactt cgagtatttg atcccggcca atgccgatcctgttgttact acacagaata 3120 ttatcgttac aggacagggt gaagttgtaa tccccggtggtgtttacgac tattgcatta 3180 cgaacccgga acctgcatcc ggaaagatgt ggatcgcaggagatggaggc aaccagcctg 3240 cacgttatga cgatttcaca ttcgaagcag gcaagaagtacaccttcacg atgcgtcgcg 3300 ccggaatggg agatggaact gatatggaag tcgaagacgattcacctgca agctatacct 3360 acacggtgta tcgtgacggc acgaagatca aggaaggtctgacagctacg acattcgaag 3420 aagacggtgt agctgcaggc aatcatgagt attgcgtggaagttaagtac acagccggcg 3480 tatctccgaa ggtatgtaaa gacgttacgg tagaaggatccaatgaattt gctcctgtac 3540 agaacctgac cggtagttca gtaggtcaga aagtaacgcttaagtgggat gcacctaatg 3600 gtaccccgaa tccgaatcca aatccgaatc cgaatccgggaacaacactt tccgaatcat 3660 tcgaaaatgg tattccggca tcttggaaga cgatcgatgcagacggtgac gggcatggct 3720 ggaaacctgg aaatgctccc ggaatcgctg gctacaatagcaatggttgt gtatattcag 3780 agtcattcgg tcttggtggt ataggagttc ttacccctgacaactatctg ataacaccgg 3840 cattggattt gcctaacgga ggtaagttga ctttctgggtatgcgcacag gatgctaatt 3900 atgcatccga gcactatgcg gtgtatgcat cttcgaccggtaacgatgca tccaacttca 3960 cgaatgcttt gttggaagag acgattacgg caaaaggtgttcgctcgccg aaagctattc 4020 gtggtcgtat acagggtact tggcgccaga agacggtagaccttcccgca ggtacgaaat 4080 atgttgcttt ccgtcacttc caaagcacgg atatgttctacatcgacctt gatgaggttg 4140 agatcaaggc caatggcaag cgcgcagact tcacggaaacgttcgagtct tctactcatg 4200 gagaggcacc agcggaatgg actactatcg atgccgatggcgatggtcag ggttggctct 4260 gtctgtcttc cggacaattg gactggctga cagctcatggcggcagcaac gtagtaagct 4320 ctttctcatg gaatggaatg gctttgaatc ctgataactatctcatctca aaggatgtta 4380 caggcgcaac gaaggtaaag tactactatg cagtcaacgacggttttccc ggggatcact 4440 atgcggtgat gatctccaag acgggcacga acgccggagacttcacggtt gttttcgaag 4500 aaacgcctaa cggaataaat aagggcggag caagattcggtctttccacg gaagccaatg 4560 gcgccaaacc tcaaagtgta tggatcgagc gtacggtagatttgcctgca ggcacgaagt 4620 atgttgcttt ccgtcactac aattgctcgg atttgaactacattcttttg gatgatattc 4680 agttcaccat gggtggcagc cccaccccga ccgattatacctacacggtg tatcgtgatg 4740 gtacgaagat caaggaaggt ttgaccgaaa cgaccttcgaagaagacggc gtagctacgg 4800 gcaatcatga gtattgcgtg gaagtgaagt acacagccggcgtatctccg aagaaatgtg 4860 taaacgtaac tgttaattcg acacagttca atcctgtacagaacctgacg gcagaacaag 4920 ctcctaacag catggatgca atccttaaat ggaatgcaccggcatctaag cgtgcggaag 4980 ttctgaacga agacttcgaa aatggtattc ctgcctcatggaagacgatc gatgcagacg 5040 gtgacggcaa caattggacg acgacccctc ctcccggaggctcctctttt gcaggtcaca 5100 acagtgcgat ctgtgtctct tcagcttctt atatcaactttgaaggtcct cagaaccctg 5160 ataactatct ggttacaccg gagctttctc ttcctggcggaggaacgctt actttctggg 5220 tatgtgcaca agatgccaat tatgcatcag agcactatgccgtgtacgca tcttctacgg 5280 gtaacgacgc ttccaacttc gccaacgctt tgttggaagaagtgctgacg gccaagacag 5340 ttgttacggc acctgaagcc attcgtggta ctcgtgctcagggcacctgg tatcaaaaga 5400 cggtacagtt gcctgcgggt actaagtatg ttgccttccgtcacttcggc tgtacggact 5460 tcttctggat caaccttgat gatgttgtaa tcacttcagggaacgctccg tcttacacct 5520 atacgatcta tcgtaataat acacagatag catcaggcgtaacggagact acttaccgag 5580 atccggactt ggctaccggt ttttacacgt acggtgtaaaggttgtttac ccgaacggag 5640 aatcagctat cgaaactgct acgttgaata tcacttcgttggcagacgta acggctcaga 5700 agccttacac gctgacagtt gtaggaaaga cgatcacggtaacttgccaa ggcgaagcta 5760 tgatctacga catgaacggt cgtcgtctgg cagcgggtcgcaacacggtt gtttacacgg 5820 ctcagggcgg ccactatgca gtcatggttg tcgttgacggcaagtcttac gtagagaaac 5880 tcgctgtaaa gtaaatctgt cttggactcg gagactttgtgcagacactt ttaagatagg 5940 tctgtaattg tctcagagta tgaatcggtc gcccgacttccttaaaagga ggtcgggcga 6000 9 199 PRT Bos taurus 9 Arg Pro Lys His ProIle Lys His Gln Gly Leu Pro Gln Glu Val Leu 1 5 10 15 Asn Glu Asn LeuLeu Arg Phe Phe Val Ala Pro Phe Pro Gln Val Phe 20 25 30 Gly Lys Glu LysVal Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser 35 40 45 Thr Glu Asp GlnAla Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser 50 55 60 Ile Ser Ser SerGlu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His 65 70 75 80 Ile Gln LysGlu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu 85 90 95 Gln Leu LeuArg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val 100 105 110 Pro AsnSer Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His 115 120 125 AlaGln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr 130 135 140Phe Tyr Pro Glu Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro 145 150155 160 Ser Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala165 170 175 Pro Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn SerGlu 180 185 190 Lys Thr Thr Met Pro Leu Trp 195 10 1706 PRTPorphyromonas gingivalis 10 Met Lys Asn Leu Asn Lys Phe Val Ser Ile AlaLeu Cys Ser Ser Leu 1 5 10 15 Leu Gly Gly Met Ala Phe Ala Gln Gln ThrGlu Leu Gly Arg Asn Pro 20 25 30 Asn Val Arg Leu Leu Glu Ser Thr Gln GlnSer Val Thr Lys Val Gln 35 40 45 Phe Arg Met Asp Asn Leu Lys Phe Thr GluVal Gln Thr Pro Lys Gly 50 55 60 Ile Gly Gln Val Pro Thr Tyr Thr Glu GlyVal Asn Leu Ser Glu Lys 65 70 75 80 Gly Met Pro Thr Leu Pro Ile Leu SerArg Ser Leu Ala Val Ser Asp 85 90 95 Thr Arg Glu Met Lys Val Glu Val ValSer Ser Lys Phe Ile Glu Lys 100 105 110 Lys Asn Val Leu Ile Ala Pro SerLys Gly Met Ile Met Arg Asn Glu 115 120 125 Asp Pro Lys Lys Ile Pro TyrVal Tyr Gly Lys Thr Tyr Ser Gln Asn 130 135 140 Lys Phe Phe Pro Gly GluIle Ala Thr Leu Asp Asp Pro Phe Ile Leu 145 150 155 160 Arg Asp Val ArgGly Gln Val Val Asn Phe Ala Pro Leu Gln Tyr Asn 165 170 175 Pro Val ThrLys Thr Leu Arg Ile Tyr Thr Glu Ile Thr Val Ala Val 180 185 190 Ser GluThr Ser Glu Gln Gly Lys Asn Ile Leu Asn Lys Lys Gly Thr 195 200 205 PheAla Gly Phe Glu Asp Thr Tyr Lys Arg Met Phe Met Asn Tyr Glu 210 215 220Pro Gly Arg Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Arg Met Ile 225 230235 240 Val Ile Val Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Phe Val Asp245 250 255 Trp Lys Asn Gln Arg Gly Leu Arg Thr Glu Val Lys Val Ala GluAsp 260 265 270 Ile Ala Ser Pro Val Thr Ala Asn Ala Ile Gln Gln Phe ValLys Gln 275 280 285 Glu Tyr Glu Lys Glu Gly Asn Asp Leu Thr Tyr Val LeuLeu Ile Gly 290 295 300 Asp His Lys Asp Ile Pro Ala Lys Ile Thr Pro GlyIle Lys Ser Asp 305 310 315 320 Gln Val Tyr Gly Gln Ile Val Gly Asn AspHis Tyr Asn Glu Val Phe 325 330 335 Ile Gly Arg Phe Ser Cys Glu Ser LysGlu Asp Leu Lys Thr Gln Ile 340 345 350 Asp Arg Thr Ile His Tyr Glu ArgAsn Ile Thr Thr Glu Asp Lys Trp 355 360 365 Leu Gly Gln Ala Leu Cys IleAla Ser Ala Glu Gly Gly Pro Ser Ala 370 375 380 Asp Asn Gly Glu Ser AspIle Gln His Glu Asn Val Ile Ala Asn Leu 385 390 395 400 Leu Thr Gln TyrGly Tyr Thr Lys Ile Ile Lys Cys Tyr Asp Pro Gly 405 410 415 Val Thr ProLys Asn Ile Ile Asp Ala Phe Asn Gly Gly Ile Ser Leu 420 425 430 Ala AsnTyr Thr Gly His Gly Ser Glu Thr Ala Trp Gly Thr Ser His 435 440 445 PheGly Thr Thr His Val Lys Gln Leu Thr Asn Ser Asn Gln Leu Pro 450 455 460Phe Ile Phe Asp Val Ala Cys Val Asn Gly Asp Phe Leu Phe Ser Met 465 470475 480 Pro Cys Phe Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gly Lys Pro485 490 495 Thr Gly Thr Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser TrpAla 500 505 510 Ser Pro Met Arg Gly Gln Asp Glu Met Asn Glu Ile Leu CysGlu Lys 515 520 525 His Pro Asn Asn Ile Lys Arg Thr Phe Gly Gly Val ThrMet Asn Gly 530 535 540 Met Phe Ala Met Val Glu Lys Tyr Lys Lys Asp GlyGlu Lys Met Leu 545 550 555 560 Asp Thr Trp Thr Val Phe Gly Asp Pro SerLeu Leu Val Arg Thr Leu 565 570 575 Val Pro Thr Lys Met Gln Val Thr AlaPro Ala Gln Ile Asn Leu Thr 580 585 590 Asp Ala Ser Val Asn Val Ser CysAsp Tyr Asn Gly Ala Ile Ala Thr 595 600 605 Ile Ser Ala Asn Gly Lys MetPhe Gly Ser Ala Val Val Glu Asn Gly 610 615 620 Thr Ala Thr Ile Asn LeuThr Gly Leu Thr Asn Glu Ser Thr Leu Thr 625 630 635 640 Leu Thr Val ValGly Tyr Asn Lys Glu Thr Val Ile Lys Thr Ile Asn 645 650 655 Thr Asn GlyGlu Pro Asn Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala 660 665 670 Thr ThrGln Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pro Ser Thr 675 680 685 LysThr Asn Ala Thr Thr Asn Thr Ala Arg Ser Val Asp Gly Ile Arg 690 695 700Glu Leu Val Leu Leu Ser Val Ser Asp Ala Pro Glu Leu Leu Arg Ser 705 710715 720 Gly Gln Ala Glu Ile Val Leu Glu Ala His Asp Val Trp Asn Asp Gly725 730 735 Ser Gly Tyr Gln Ile Leu Leu Asp Ala Asp His Asp Gln Tyr GlyGln 740 745 750 Val Ile Pro Ser Asp Thr His Thr Leu Trp Pro Asn Cys SerVal Pro 755 760 765 Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Val Pro GluAsn Ala Asp 770 775 780 Pro Ser Cys Ser Pro Thr Asn Met Ile Met Asp GlyThr Ala Ser Val 785 790 795 800 Asn Ile Pro Ala Gly Thr Tyr Asp Phe AlaIle Ala Ala Pro Gln Ala 805 810 815 Asn Ala Lys Ile Trp Ile Ala Gly GlnGly Pro Thr Lys Glu Asp Asp 820 825 830 Tyr Val Phe Glu Ala Gly Lys LysTyr His Phe Leu Met Lys Lys Met 835 840 845 Gly Ser Gly Asp Gly Thr GluLeu Thr Ile Ser Glu Gly Gly Gly Ser 850 855 860 Asp Tyr Thr Tyr Thr ValTyr Arg Asp Gly Thr Lys Ile Lys Glu Gly 865 870 875 880 Leu Thr Ala ThrThr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His 885 890 895 Glu Tyr CysVal Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Val 900 905 910 Cys LysAsp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val Gln 915 920 925 AsnLeu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp Asp 930 935 940Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro 945 950955 960 Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile Pro965 970 975 Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly TrpLys 980 985 990 Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn GlyCys Val 995 1000 1005 Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly ValLeu Thr Pro Asp 1010 1015 1020 Asn Tyr Leu Ile Thr Pro Ala Leu Asp LeuPro Asn Gly Gly Lys Leu 1025 1030 1035 1040 Thr Phe Trp Val Cys Ala GlnAsp Ala Asn Tyr Ala Ser Glu His Tyr 1045 1050 1055 Ala Val Tyr Ala SerSer Thr Gly Asn Asp Ala Ser Asn Phe Thr Asn 1060 1065 1070 Ala Leu LeuGlu Glu Thr Ile Thr Ala Lys Gly Val Arg Ser Pro Glu 1075 1080 1085 AlaMet Arg Gly Arg Ile Gln Gly Thr Trp Arg Gln Lys Thr Val Asp 1090 10951100 Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg His Phe Gln Ser Thr1105 1110 1115 1120 Asp Met Phe Tyr Ile Asp Leu Asp Glu Val Glu Ile LysAla Asn Gly 1125 1130 1135 Lys Arg Ala Asp Phe Thr Glu Thr Phe Glu SerSer Thr His Gly Glu 1140 1145 1150 Ala Pro Ala Glu Trp Thr Thr Ile AspAla Asp Gly Asp Gly Gln Gly 1155 1160 1165 Trp Leu Cys Leu Ser Ser GlyGln Leu Asp Trp Leu Thr Ala His Gly 1170 1175 1180 Gly Thr Asn Val ValSer Ser Phe Ser Trp Asn Gly Met Ala Leu Asn 1185 1190 1195 1200 Pro AspAsn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val 1205 1210 1215Lys Tyr Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala 12201225 1230 Val Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr ValVal 1235 1240 1245 Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly AlaArg Phe Gly 1250 1255 1260 Leu Ser Thr Glu Ala Asp Gly Ala Lys Pro GlnSer Val Trp Ile Glu 1265 1270 1275 1280 Arg Thr Val Asp Leu Pro Ala GlyThr Lys Tyr Val Ala Phe Arg His 1285 1290 1295 Tyr Asn Cys Ser Asp LeuAsn Tyr Ile Leu Leu Asp Asp Ile Gln Phe 1300 1305 1310 Thr Met Gly GlySer Pro Thr Pro Thr Asp Tyr Thr Tyr Thr Val Tyr 1315 1320 1325 Arg AspGly Thr Lys Ile Lys Glu Gly Leu Thr Glu Thr Thr Phe Glu 1330 1335 1340Glu Asp Gly Val Ala Thr Gly Asn His Glu Tyr Cys Val Glu Val Lys 13451350 1355 1360 Tyr Thr Ala Gly Val Ser Pro Lys Lys Cys Val Asn Val ThrVal Asn 1365 1370 1375 Ser Thr Gln Phe Asn Pro Val Lys Asn Leu Lys AlaGln Pro Asp Gly 1380 1385 1390 Gly Asp Val Val Leu Lys Trp Glu Ala ProSer Ala Lys Lys Thr Glu 1395 1400 1405 Gly Ser Arg Glu Val Lys Arg IleGly Asp Gly Leu Phe Val Thr Ile 1410 1415 1420 Glu Pro Ala Asn Asp ValArg Ala Asn Glu Ala Lys Val Val Leu Ala 1425 1430 1435 1440 Ala Asp AsnVal Trp Gly Asp Asn Thr Gly Tyr Gln Phe Leu Leu Asp 1445 1450 1455 AlaAsp His Asn Thr Phe Gly Ser Val Ile Pro Ala Thr Gly Pro Leu 1460 14651470 Phe Thr Gly Thr Ala Ser Ser Asp Leu Tyr Ser Ala Asn Phe Glu Ser1475 1480 1485 Leu Ile Pro Ala Asn Ala Asp Pro Val Val Thr Thr Gln AsnIle Ile 1490 1495 1500 Val Thr Gly Gln Gly Glu Val Val Ile Pro Gly GlyVal Tyr Asp Tyr 1505 1510 1515 1520 Cys Ile Thr Asn Pro Glu Pro Ala SerGly Lys Met Trp Ile Ala Gly 1525 1530 1535 Asp Gly Gly Asn Gln Pro AlaArg Tyr Asp Asp Phe Thr Phe Glu Ala 1540 1545 1550 Gly Lys Lys Tyr ThrPhe Thr Met Arg Arg Ala Gly Met Gly Asp Gly 1555 1560 1565 Thr Asp MetGlu Val Glu Asp Asp Ser Pro Ala Ser Tyr Thr Tyr Thr 1570 1575 1580 ValTyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu Thr Glu Thr Thr 1585 15901595 1600 Tyr Arg Asp Ala Gly Met Ser Ala Gln Ser His Glu Tyr Cys ValGlu 1605 1610 1615 Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Val Cys ValAsp Tyr Ile 1620 1625 1630 Pro Asp Gly Val Ala Asp Val Thr Ala Gln LysPro Tyr Thr Leu Thr 1635 1640 1645 Val Val Gly Lys Thr Ile Thr Val ThrCys Gln Gly Glu Ala Met Ile 1650 1655 1660 Tyr Asp Met Asn Gly Arg ArgLeu Ala Ala Gly Arg Asn Thr Val Val 1665 1670 1675 1680 Tyr Thr Ala GlnGly Gly Tyr Tyr Ala Val Met Val Val Val Asp Gly 1685 1690 1695 Lys SerTyr Val Glu Lys Leu Ala Ile Lys 1700 1705 11 1732 PRT Porphyromonasgingivalis 11 Met Arg Lys Leu Leu Leu Leu Ile Ala Ala Ser Leu Leu GlyVal Gly 1 5 10 15 Leu Tyr Ala Gln Ser Ala Lys Ile Lys Leu Asp Ala ProThr Thr Arg 20 25 30 Thr Thr Cys Thr Asn Asn Ser Phe Lys Gln Phe Asp AlaSer Phe Ser 35 40 45 Phe Asn Glu Val Glu Leu Thr Lys Val Glu Thr Lys GlyGly Thr Phe 50 55 60 Ala Ser Val Ser Ile Pro Gly Ala Phe Pro Thr Gly GluVal Gly Ser 65 70 75 80 Pro Glu Val Pro Ala Val Arg Lys Leu Ile Ala ValPro Val Gly Ala 85 90 95 Thr Pro Val Val Arg Val Lys Ser Phe Thr Glu GlnVal Tyr Ser Leu 100 105 110 Asn Gln Tyr Gly Ser Glu Lys Leu Met Pro HisGln Pro Ser Met Ser 115 120 125 Lys Ser Asp Asp Pro Glu Lys Val Pro PheVal Tyr Asn Ala Ala Ala 130 135 140 Tyr Ala Arg Lys Gly Phe Val Gly GlnGlu Leu Thr Gln Val Glu Met 145 150 155 160 Leu Gly Thr Met Arg Gly ValArg Ile Ala Ala Leu Thr Ile Asn Pro 165 170 175 Val Gln Tyr Asp Val ValAla Asn Gln Leu Lys Val Arg Asn Asn Ile 180 185 190 Glu Ile Glu Val SerPhe Gln Gly Ala Asp Glu Val Ala Thr Gln Arg 195 200 205 Leu Tyr Asp AlaSer Phe Ser Pro Tyr Phe Glu Thr Ala Tyr Lys Gln 210 215 220 Leu Phe AsnArg Asp Val Tyr Thr Asp His Gly Asp Leu Tyr Asn Thr 225 230 235 240 ProVal Arg Met Leu Val Val Ala Gly Ala Lys Phe Lys Glu Ala Leu 245 250 255Lys Pro Trp Leu Thr Trp Lys Ala Gln Lys Gly Phe Tyr Leu Asp Val 260 265270 His Tyr Thr Asp Glu Ala Glu Val Gly Thr Thr Asn Ala Ser Ile Lys 275280 285 Ala Phe Ile His Lys Lys Tyr Asn Asp Gly Leu Ala Ala Ser Ala Ala290 295 300 Pro Val Phe Leu Ala Leu Val Gly Asp Thr Asp Val Ile Ser GlyGlu 305 310 315 320 Lys Gly Lys Lys Thr Lys Lys Val Thr Asp Leu Tyr TyrSer Ala Val 325 330 335 Asp Gly Asp Tyr Phe Pro Glu Met Tyr Thr Phe ArgMet Ser Ala Ser 340 345 350 Ser Pro Glu Glu Leu Thr Asn Ile Ile Asp LysVal Leu Met Tyr Glu 355 360 365 Lys Ala Thr Met Pro Asp Lys Ser Tyr LeuGlu Lys Val Leu Leu Ile 370 375 380 Ala Gly Ala Asp Tyr Ser Trp Asn SerGln Val Gly Gln Pro Thr Ile 385 390 395 400 Lys Tyr Gly Met Gln Tyr TyrTyr Asn Gln Glu His Gly Tyr Thr Asp 405 410 415 Val Tyr Asn Tyr Leu LysAla Pro Tyr Thr Gly Cys Tyr Ser His Leu 420 425 430 Asn Thr Gly Val SerPhe Ala Asn Tyr Thr Ala His Gly Ser Glu Thr 435 440 445 Ala Trp Ala AspPro Leu Leu Thr Thr Ser Gln Leu Lys Ala Leu Thr 450 455 460 Asn Lys AspLys Tyr Phe Leu Ala Ile Gly Asn Cys Cys Ile Thr Ala 465 470 475 480 GlnPhe Asp Tyr Val Gln Pro Cys Phe Gly Glu Val Ile Thr Arg Val 485 490 495Lys Glu Lys Gly Ala Tyr Ala Tyr Ile Gly Ser Ser Pro Asn Ser Tyr 500 505510 Trp Gly Glu Asp Tyr Tyr Trp Ser Val Gly Ala Asn Ala Val Phe Gly 515520 525 Val Gln Pro Thr Phe Glu Gly Thr Ser Met Gly Ser Tyr Asp Ala Thr530 535 540 Phe Leu Glu Asp Ser Tyr Asn Thr Val Asn Ser Ile Met Trp AlaGly 545 550 555 560 Asn Leu Ala Ala Thr His Ala Gly Asn Ile Gly Asn IleThr His Ile 565 570 575 Gly Ala His Tyr Tyr Trp Glu Ala Tyr His Val LeuGly Asp Gly Ser 580 585 590 Val Met Pro Tyr Arg Ala Met Pro Lys Thr AsnThr Tyr Thr Leu Pro 595 600 605 Ala Ser Leu Pro Gln Asn Gln Ala Ser TyrSer Ile Gln Ala Ser Ala 610 615 620 Gly Ser Tyr Val Ala Ile Ser Lys AspGly Val Leu Tyr Gly Thr Gly 625 630 635 640 Val Ala Asn Ala Ser Gly ValAla Thr Val Ser Met Thr Lys Gln Ile 645 650 655 Thr Glu Asn Gly Asn TyrAsp Val Val Ile Thr Arg Ser Asn Tyr Leu 660 665 670 Pro Val Ile Lys GlnIle Gln Val Gly Glu Pro Ser Pro Tyr Gln Pro 675 680 685 Val Ser Asn LeuThr Ala Thr Thr Gln Gly Gln Lys Val Thr Leu Lys 690 695 700 Trp Glu AlaPro Ser Ala Lys Lys Ala Glu Gly Ser Arg Glu Val Lys 705 710 715 720 ArgIle Gly Asp Gly Leu Phe Val Thr Ile Glu Pro Ala Asn Asp Val 725 730 735Arg Ala Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn Val Trp Gly 740 745750 Asp Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp His Asn Thr Phe 755760 765 Gly Ser Val Ile Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala Ser770 775 780 Ser Asn Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro Ala AsnAla 785 790 795 800 Asp Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr GlyGln Gly Glu 805 810 815 Val Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys IleThr Asn Pro Glu 820 825 830 Pro Ala Ser Gly Lys Met Trp Ile Ala Gly AspGly Gly Asn Gln Pro 835 840 845 Ala Arg Tyr Asp Asp Phe Thr Phe Glu AlaGly Lys Lys Tyr Thr Phe 850 855 860 Thr Met Arg Arg Ala Gly Met Gly AspGly Thr Asp Met Glu Val Glu 865 870 875 880 Asp Asp Ser Pro Ala Ser TyrThr Tyr Thr Val Tyr Arg Asp Gly Thr 885 890 895 Lys Ile Lys Glu Gly LeuThr Ala Thr Thr Phe Glu Glu Asp Gly Val 900 905 910 Ala Ala Gly Asn HisGlu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly 915 920 925 Val Ser Pro LysVal Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu 930 935 940 Phe Ala ProVal Gln Asn Leu Thr Gly Ser Ser Val Gly Gln Lys Val 945 950 955 960 ThrLeu Lys Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn 965 970 975Pro Asn Pro Asn Pro Gly Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly 980 985990 Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly 9951000 1005 Trp Lys Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser AsnGly 1010 1015 1020 Cys Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile GlyVal Leu Thr 1025 1030 1035 1040 Pro Asp Asn Tyr Leu Ile Thr Pro Ala LeuAsp Leu Pro Asn Gly Gly 1045 1050 1055 Lys Leu Thr Phe Trp Val Cys AlaGln Asp Ala Asn Tyr Ala Ser Glu 1060 1065 1070 His Tyr Ala Val Tyr AlaSer Ser Thr Gly Asn Asp Ala Ser Asn Phe 1075 1080 1085 Thr Asn Ala LeuLeu Glu Glu Thr Ile Thr Ala Lys Gly Val Arg Ser 1090 1095 1100 Pro LysAla Ile Arg Gly Arg Ile Gln Gly Thr Trp Arg Gln Lys Thr 1105 1110 11151120 Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg His Phe Gln1125 1130 1135 Ser Thr Asp Met Phe Tyr Ile Asp Leu Asp Glu Val Glu IleLys Ala 1140 1145 1150 Asn Gly Lys Arg Ala Asp Phe Thr Glu Thr Phe GluSer Ser Thr His 1155 1160 1165 Gly Glu Ala Pro Ala Glu Trp Thr Thr IleAsp Ala Asp Gly Asp Gly 1170 1175 1180 Gln Gly Trp Leu Cys Leu Ser SerGly Gln Leu Asp Trp Leu Thr Ala 1185 1190 1195 1200 His Gly Gly Ser AsnVal Val Ser Ser Phe Ser Trp Asn Gly Met Ala 1205 1210 1215 Leu Asn ProAsp Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr 1220 1225 1230 LysVal Lys Tyr Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His 1235 12401245 Tyr Ala Val Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr1250 1255 1260 Val Val Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly GlyAla Arg 1265 1270 1275 1280 Phe Gly Leu Ser Thr Glu Ala Asn Gly Ala LysPro Gln Ser Val Trp 1285 1290 1295 Ile Glu Arg Thr Val Asp Leu Pro AlaGly Thr Lys Tyr Val Ala Phe 1300 1305 1310 Arg His Tyr Asn Cys Ser AspLeu Asn Tyr Ile Leu Leu Asp Asp Ile 1315 1320 1325 Gln Phe Thr Met GlyGly Ser Pro Thr Pro Thr Asp Tyr Thr Tyr Thr 1330 1335 1340 Val Tyr ArgAsp Gly Thr Lys Ile Lys Glu Gly Leu Thr Glu Thr Thr 1345 1350 1355 1360Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His Glu Tyr Cys Val Glu 13651370 1375 Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Lys Cys Val Asn ValThr 1380 1385 1390 Val Asn Ser Thr Gln Phe Asn Pro Val Gln Asn Leu ThrAla Glu Gln 1395 1400 1405 Ala Pro Asn Ser Met Asp Ala Ile Leu Lys TrpAsn Ala Pro Ala Ser 1410 1415 1420 Lys Arg Ala Glu Val Leu Asn Glu AspPhe Glu Asn Gly Ile Pro Ala 1425 1430 1435 1440 Ser Trp Lys Thr Ile AspAla Asp Gly Asp Gly Asn Asn Trp Thr Thr 1445 1450 1455 Thr Pro Pro ProGly Gly Ser Ser Phe Ala Gly His Asn Ser Ala Ile 1460 1465 1470 Cys ValSer Ser Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro 1475 1480 1485Asp Asn Tyr Leu Val Thr Pro Glu Leu Ser Leu Pro Gly Gly Gly Thr 14901495 1500 Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser GluHis 1505 1510 1515 1520 Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp AlaSer Asn Phe Ala 1525 1530 1535 Asn Ala Leu Leu Glu Glu Val Leu Thr AlaLys Thr Val Val Thr Ala 1540 1545 1550 Pro Glu Ala Ile Arg Gly Thr ArgAla Gln Gly Thr Trp Tyr Gln Lys 1555 1560 1565 Thr Val Gln Leu Pro AlaGly Thr Lys Tyr Val Ala Phe Arg His Phe 1570 1575 1580 Gly Cys Thr AspPhe Phe Trp Ile Asn Leu Asp Asp Val Val Ile Thr 1585 1590 1595 1600 SerGly Asn Ala Pro Ser Tyr Thr Tyr Thr Ile Tyr Arg Asn Asn Thr 1605 16101615 Gln Ile Ala Ser Gly Val Thr Glu Thr Thr Tyr Arg Asp Pro Asp Leu1620 1625 1630 Ala Thr Gly Phe Tyr Thr Tyr Gly Val Lys Val Val Tyr ProAsn Gly 1635 1640 1645 Glu Ser Ala Ile Glu Thr Ala Thr Leu Asn Ile ThrSer Leu Ala Asp 1650 1655 1660 Val Thr Ala Gln Lys Pro Tyr Thr Leu ThrVal Val Gly Lys Thr Ile 1665 1670 1675 1680 Thr Val Thr Cys Gln Gly GluAla Met Ile Tyr Asp Met Asn Gly Arg 1685 1690 1695 Arg Leu Ala Ala GlyArg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly 1700 1705 1710 His Tyr AlaVal Met Val Val Val Asp Gly Lys Ser Tyr Val Glu Lys 1715 1720 1725 LeuAla Val Lys 1730 12 7 PRT Porphyromonas gingivalis 12 Tyr Glu Gly AspIle Lys Asp 1 5 13 9 PRT Porphyromonas gingivalis 13 Lys Asp Phe Val AspTrp Lys Asn Gln 1 5 14 8 PRT Porphyromonas gingivalis 14 Asp Val Tyr ThrAsp His Gly Asp 1 5 15 6 PRT Porphyromonas gingivalis 15 Thr His Ile GlyAla His 1 5

What is claimed is:
 1. A purified antigenic complex for use in raisingan antibody response directed against Porphyromonas gingivalis, thecomplex comprising at least one multimeric protein complex ofarginine-specific and lysine-specific thiol endopeptidases eachcontaining at least one adhesion domain, the complex having a molecularweight of about 294 to about 323 kDa wherein the multimeric proteincomplex comprises 9 proteins, the 9 proteins having the followingN-terminal sequences: DVYTDHGDLYNTPVRML (SEQ ID NO:1),YTPVEEKQNGRMIVIVAKKYEGD (SEQ ID NO:2),SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHFL (SEQ ID NO:3),PQSVWIERTVDLPAGTKYVAFR (SEQ ID NO:4), ANEAKVVLAADNVWGNTGYQFLLDA (SEQ IDNO:5), ANEAKVVLAADNVWGDNTGYQFLLDA SEQ ID NO:5), PQSVWIERTVDLPAGTKYVAFR(SEQ ID NO:4), ADFTETFESSTHGEAPAEWTTIDA (SEQ ID NO:6), andADFTETFESSTHGEAPAEWTTIDA (SEQ ID NO:6).
 2. A purified antigenic complexas claimed in claim 1 in which the multimeric protein complex isassociated with virulent strains of Porphyromonas gingivalis.
 3. Apurified antigenic complex as claimed in claim 1 in which the 9 proteinsare PrtK48, PrtR45, PrtR44, PrtK39, PrtK44, PrtR27, PrtR17, PrtK15 andPrtR15.
 4. A purified antigenic complex as claimed in claim 1 in whichthe thiol endopeptidases are rendered inactive.
 5. A purified antigeniccomplex as claimed in claim 4 in which the thiol endopeptidases arerendered inactive by oxidation.
 6. A purified antigenic complex asclaimed in claim 4 in which the thiol endopeptidases are renderedinactive by mutation.
 7. A purified antigenic complex as claimed inclaim 1 in which the multimeric protein complex is encoded by the DNAsequence shown in FIGS. 8B (SEQ ID NO:7) and 9B (SEQ ID NO.: 8).
 8. Acomposition for use in eliciting an immune response directed againstPorphyromonas gingivalis, the composition comprising an effective amountof the complex as claimed in claim 1 and a suitable adjuvant and/oracceptable carrier.
 9. A method of reducing the prospect of P.gingivalis infection in an individual and/or severity of disease, themethod comprising administering to the individual an amount of thecomposition as claimed in claim 8 effective to induce an immune responsein the individual directed against P. gingivalis.